April O'Neil is a reporter for Channel 6 news in New York who has been researching a gang called the Foot Clan which has been terrorizing the city. She questions a dock worker about shipments of chemicals that may be linked to the Foot Clan. April eventually learns that something is being brought in by the docks. That night, she returns and sees the Foot Clan unloading cargo. April tries to record footage using her phone, but a shadowy figure arrives and takes out the Foot Soldiers one by one. She tells her coworkers and her boss Bernadette Thompson, but no one believes her story.

The Foot Clan next attacks a subway station. April rushes to the scene, hoping to encounter the vigilante or find evidence confirming his existence. She sees four figures this time, who disappear after defeating the Foot Clan. She follows them to a rooftop and tries to photograph them. The Turtles Leonardo, Michelangelo, Donatello, and Raphael notice her and delete the camera's images, warning her not to divulge their existence. She asks them who they are as they leave, and they say, "Teenage Mutant Ninja Turtles."

April runs home and opens a box filled with documents, pictures, and July 1999 videos on "Project Renaissance", which involved her now-deceased father. She notices that the turtles she cared for from her father's laboratory fifteen years earlier seem similar to the Ninja Turtles. She recalls that her father was developing some type of mutagen. She continues researching and eventually realizes that the Ninja Turtles are the turtles from the laboratory.

Once again, she tries to convince Bernadette Thompson that the Turtles are real. Bernadette becomes so infuriated that she fires April. April tries telling her cameraman Vern Fenwick about them, but he does not believe her either. He does agree to take her to the old laboratory, though. There she finds her father's lab partner Eric Sacks.

April tells him about the Ninja Turtles, showing him the photos. Sacks explains Project Renaissance: he and her father were cultivating the mutagen for its healing properties. Sacks theorizes that, when the laboratory was destroyed, the mutagen must have somehow caused the turtles to mutate into humanoids.

Meanwhile in the sewers, the Turtles tell their master, Splinter, that April has spotted them. Splinter orders them to find April and bring her to their lair, as she is now in great danger from the Foot Clan since she made contact with the Turtles.

The Turtles find April and blindfold her so she won't know their lair's location. They take her to Splinter, who explains how she saved their lives years before when she rescued them from the fire and released them into the sewers. April admits that she has told her father's associate about them, unaware that Sacks is actually the adoptive son of the Foot Clan's leader The Shredder.

Meanwhile, Sacks relays the information to Shredder. Shredder and Sacks plan to spread a deadly virus throughout New York, causing a quarantine, in order to seize control by offering the mutagen as a cure. Shredder needs the Turtles to extract the mutagen from their blood.

He and his gang find Splinter and the Turtles in the sewers, and a battle ensues. They are overwhelmed and Shredder captures Leo, Donnie and Mikey and leaves Splinter severely injured. Thought dead, Raphael survived the wreckage of the lair. Splinter instructs Raphael and April to save the other three Turtles before Shredder can extract the mutagen from them. April calls Vern to give them a ride to the laboratory where the other three Turtles are being held.

When they arrive, April frees the Turtles, who join Raphael in fighting Shredder, but Shredder escapes. April, the Turtles, and Vern escape down a snowy mountain with The Foot and Karai in pursuit, and manage to get away.

The Turtles plan to attack Shredder on the rooftop of Sacks' building before he is able to release the toxin, while April and Vern search for the mutagen and battle Sacks inside the building where Sacks is knocked out by Vern. April finds the mutagen and heads onto the rooftop to give it to the Turtles. With April's help, the Turtles finally defeat Shredder who falls off the roof where he is immediately surrounded by the police. He is last seen touching the spilled mutagen.

That night, Vern attempts to impress April with a Ford Crown Victoria Police Interceptor, but unfortunately fails when the Turtles come in with a vehicle of their own and accidentally blow it up with an RPG. The turtles then offer April a ride home but she kindly turns down the offer. The film ends with Mikey serenading April with "Happy Together" a love song performed by the 60's rock band The Turtles.