The Mystery Inc. gang meet with their friend, Eric Staufer, a computer science major at the university, who created a laser that has the ability to send people into cyberspace. A video game featuring Scooby and the gang was designed to test the laser, but during the test, a Phantom Virus has escaped from cyberspace, and begins wreaking havoc in the university. Can Mystery Inc. stop the Phantom Virus, and find out who is responsible for releasing it?

In Scooby-Doo and the Cyber Chase for Gameboy Advance, you must discover the identity of the suspect responsible for unleashing the Phantom Virus. This is done by navigating seven levels; the main lab level, which contains CDs, grant access to six additional levels, which may only be reached via the computer in cyberspace, and the laser in the robotics lab section of the main level sends a character there. Each member of the Mystery Inc. gang is available, but only one character may play at a time. Only two CDs are available at the start. Two additional CDs are gained upon completion of a computer level, which appear in the main lab level in the Computer Lab room, and when picked up unlocks more computer levels in the game to complete. You cannot enter the computer levels unless you pick up the CDs from the main lab level.

Each level consists of various pick-ups: disguises, which helps you sneak past the various monsters on each level, smiley faces, which increases your character's health, Scooby Snacks, including a counter telling you how many are needed to finish the level, and a Scooby Snacks Box, which appears after picking up enough Scooby Snacks, allowing you to successfully complete the level. Level bosses either contain classic Scooby villains, or the Phantom Virus; the villains must be defeated to complete the level, the Phantom Virus cannot be defeated until the final level, and must be avoided until you pick up the Scooby Snacks Box. Contact with monsters, villains and the Phantom Virus' electric bolts will reduce your character's health; if a character's health is gone, that character is captured in cyberspace, and another character will have to try the level.

Points are earned for collecting Scooby Snacks, and evading the monsters. Clues are also collected during the game; the game saves via a password system, which records your collected clues, turns (remaining characters), and your score after each level. After finishing the final level, the game will ask you which of the four suspects in the game was responsible for unleashing the Phantom Virus. You win the game if you correctly identify the suspect who released the Phantom Virus based on the clues you've collected during the game.