Chronologically, E-100's first appearance was in Amy Rose's story of Sonic Adventure. He was sent by Doctor Robotnik to capture a bird known as a Flicky, which had a Chaos Emerald. Amy Rose decided to take care of the bird (who she unoriginally dubs "Birdy"), but ZERO arrived and chased Amy, but she evaded the robot by hiding in a restaurant. Alpha then tracks down the bird, who is accompanied by not only Amy, but also his creators arch-enemy, Sonic the Hedgehog. Amy enters the nearby Twinkle Park, with both Sonic and ZERO in pursuit. Although it fails to capture the bird and hedgehog in the Twinkle Park, he does manage to capture them as they exit the park and it takes them to the Mystic Ruins, with Sonic tailing them. However, ZERO is beamed up into the floating fortress, the Egg Carrier, where he delivers his captives to a prison cell. After being freed by a malfunctioning E-Series robot, E-102, Amy Rose escapes into the Hot Shelter portion of the Egg Carrier, where Alpha again gives chase. He fails, but Eggman still manages to get Birdy's Chaos Emerald when they get to the surface of the flying battleship. Alpha, even though Birdy doesn't have the powerful gem anymore, Alpha still chases it when Amy as she traverses through another of Robotnik's bases, the Final Egg. Then, on the crashed Egg Carrier, Alpha hits and knocks Birdy to the ground as it reunites with it's family. Amy then goes into the rage and destroys E-100 by knocking it into an electrical gate with her massive Piko Piko Hammer, and then hitting its core.
During the gameplay, Alpha will constantly chase you throughout all of Amy's levels. He will smash through walls, windows mirrors and crates to get you. He can shoot his fists at you to harm you and kill you if you have no rings. You can stun him temporarily by hitting him with your hammer and he can't chase you at certain points. However, you cannot kill him during normal play, unlike in the boss fight.