Resident Evil Zero is a third-person survival horror video game. The gameplay remains largely the same as previous entries in the series, however there are two significant differences.

First is the "partner zapping" system. Instead of choosing one of two player characters at the beginning, there are two protagonists to be controlled throughout the entire game. The player may switch between police officer and medic Rebecca Chambers and convicted ex-soldier Billy Coen. If they travel together, either one of them can be controlled while the other character is handled by the game's AI. The player may also control both simultaneously or split them up entirely to explore areas separately. Each character has unique abilities. Rebecca has a mixing kit which allows her to combine herbs and other chemicals, but she is weak defensively. In contrast, Billy can move heavy objects and has higher defense. The partner system is central to solving the game's puzzles.