Fifteen years ago, a Techno Cosmic Research Institute strike force is ordered by executive Cynthia Utrom to capture rogue scientist Baxter Stockman, who stole company secrets and technology, and any research. Desiring a family, Stockman creates a mutagen to create his own mutant family, starting with a housefly maggot. Before he can create more mutagen, the strike force arrives. While it is dealt with by the fly mutant, an explosion in the fight destroys the lab and most of the research while killing Stockman. Additional TCRI forces arrive and collect the remainder of the surviving research, but forget the mutant and the last canister of mutagen, the latter of which falls into the sewers below.In the present, in New York City, four turtles (Michaelangelo, Leonardo, Raphael, and Donatello) have been raised by their adoptive father Splinter. While they are adept in the arts of ninjitsu, they are instructed to only leave for the surface world to get supplies for their home in the sewers.During these runs, they occasionally go and see attractions like outdoor movies, concerts, and sports, which makes them want to be on the surface more. When Splinter discovers what they have been doing, he punishes them, explaining their backstory: the mutagen from Stockman's house fell on the four baby turtles, who were found by Splinter (then a rat that was cynical and untrusting of the surface world, specifically of humans). While handling the turtles, Splinter also comes into contact with the mutagen, mutating all of them to have more human-like features. As the Turtles grew older and became more curious about the surface, he decided to take them to the surface where they are all nearly killed. This leads Splinter and the Turtles to all learn martial arts for their protection.Sometime later during one of their supply runs, the Turtles accidentally get the attention of a teenage girl named April. While yelling at the turtles but not seeing them, she gets her moped stolen. The Turtles are able to get it back after defeating the group that stole it and reveal themselves to her. April shows the turtles to her high school where the group discusses their goals: the turtles want to be embraced by the surface world, while April is an aspiring journalist, who wants to move past an embarrassing viral incident where she vomited from anxiety during her school announcements. They decide to help each other out; April has been investigating a series of violent robberies on TCRI technology by a criminal named Superfly, which have led parents to call for a city-wide curfew that would cancel the school's Prom. If she records and writes an article on who Superfly is and how the Turtles would stop him, April and the Turtles would be regarded as heroes and accepted. Meanwhile, TCRI has been following the reports of the Superfly robberies. Cynthia decides to place trackers on any technology to find him.Using leads from April, the Turtles find out that Superfly is collecting a piece of technology that night in a deal under the Brooklyn Bridge. They are able to find the part and pose as the buyers, and discover that not only is Superfly a fly mutant, but also has a gang of mutants with him like Leatherhead, Mondo Gecko, Genghis Frog, Wingnut, Scumbug, Ray Fillet, and Bebop and Rocksteady. Ecstatic that there are other mutants, Superfly invites the Turtles to go on a night in the town with him and his gang, where they bond. He explains his story and plan: like the Turtles, the mutagen evolved him and the rest of his mutant gang, and he took on the role of their father figure. When he brought them to the surface, they too were nearly killed by humans, but they escaped. In their escape, Superfly nearly killed a human. Deciding that humans are all evil and would never accept them, he would create a weapon that would turn the ooze into an airborne virus, which would mutate all non-human wildlife on the planet, and make them the dominant species while enslaving humanity in the process. The Turtles try to stop them by trying to escape with the last part, but Superfly and his gang escape with it, and the Turtles are left to be captured by TCRI, who had a tracker on the part. Having followed the Turtles from a distance, April leaves to get Splinter to rescue them.At TCRI headquarters, the Turtles are slowly "milked" of their mutagen, before they are rescued by Splinter and April. Betrayed by his sons' lies and despite stating that April is the only good human, Splinter decides that they will stop Superfly before they stay in the sewers permanently. When they reach the island, Leonardo and Splinter plead to the conscious of Superfly's gang, stating that if they enslave humanity then they are no better than the humans themselves. The gang agrees and everyone fights Superfly, destroying the machine in the process.Unfortunately, the ooze enlarges and combines Superfly with other local wildlife into a bipedal whale-like creature who dubs himself "SuperDuperFly", deciding to attack New York City. The Turtles, Splinter, the Mutants, and April head to mainland New York City to stop him once and for all. While the Turtles, Splinter, and the Mutants fight, April is able to overcome her anxiety and explain on live air to the public what is going on, leading the citizens of New York to help stop SuperDuperFly. Donatello discovers that the blowhole on the back of his neck carries a massive amount of mutagen, and the Turtles are able to drop a canister of anti-ooze into SuperDuperFly's blowhole, returning him to being a regular fly.The Turtles, Splinter, April, and the Mutants are recognized as heroes by the city, and the boys reconcile with their father, who apologizes for his xenophobic behavior. Soon after, the Mutants move into the sewers with the Turtles and Splinter, and the Turtles enroll at April's high school, where they are all embraced as heroes.In a mid-credit scene, the Turtles are shown enjoying high school life: Michaelangelo is participating in the school's comedy improv team, Raphael has joined the wrestling team, Donatello has found the computer club, and Leonardo (who, throughout the film, has developed a crush on April) has decided to help her with her journalistic interests, now focused on TCRI who have mysteriously disappeared since the events of the film. While the five are later enjoying themselves at the school prom, Cynthia, who is spying on them (and with the now unmutated Superfly by her side) decides that the focus is to capture the Turtles by bringing in Shredder.