Plot hole: When April O'Neil calls Vernon Fenwick (drives the channel 6 van), he answers his phone and immediately says "O'Neil". When the scene cuts back to O'Neil, we see she called from a payphone. Fenwick would have no way to know it was her calling.
Plot hole: On the night after April meets the turtles for the first time, she has her boss come to her apartment and tries desperately to try and convince her that the turtles are real, unfortunately her boss wasn't convinced and she ends up getting fired. However, April had already taken two pictures of the turtles on her phone the night previously (before meeting the turtles and after they had left the roof and began jumping over other roof tops). She could have easily just shown the pictures to her boss and she would have been convinced the turtles were real. Even if Donatello deleted the picture she had taken before meeting the turtles, April had still taken a picture of the turtles after they left the roof and jumped from other roof tops, so she still could have easily been able to prove they exist.
Continuity mistake: Halfway through the film, when the turtles ask April to meet them on the rooftop, she is wearing a plain black top under her yellow jacket. In the next scene when the turtles have taken her to their lair, she is now wearing a light grey shirt with a symbol on it.
Continuity mistake: In the scene where the turtles are in the elevator singing MC Mikey, visible to the right of shot is the elevator's control panel and a light lights up every time they pass each floor. Just as the shot ends, the light lights up for floor 34. In the next shot, the elevator stops at floor 54.
Continuity mistake: In the scene where Raphael, April and Vernon are breaking into Eric Sacks' mansion with the Channel 6 van, the guards begin firing at the van and in one shot the windscreen is fired at and it shatters. A few seconds later, the windscreen is suddenly intact and the damage is reduced to just a few bullet holes.
Other mistake: When the turtles blood is being drained, all four tubes have blood running through them, even though there are only three turtles in the containment boxes.
Continuity mistake: When April goes to the rooftop for the first time, the ladder is a normal ladder that abruptly ends. When it cuts to her taking the photo, it is a ladder that curves over and connects to the roof, then when she is brought away from it, it briefly goes back to a typical ladder.
Continuity mistake: After the turtles, April and Vernon escape from the Sacks compound in the 18 wheeler, the truck then get stuck in the snow. When they realize they start to slide backward down the hill, Vernon guns it. But then they show the wheels spinning on the Freightliner Argosy Cabover with a single axle. Next shot goes to inside the cab and then back to outside the truck, and it's back to a dual axle truck.
Factual error: After the sewer invasion, April watches the three captured turtles drive away, then she turns to go to Splinter. Shortly thereafter, the computer screen shows GPS tracking the turtles at a location easily over an hour away from Manhattan. Likewise, the estate is referred to as being just forty minutes away from the city. (00:56:10 - 00:58:50)