Allwine was born in Glendale, California on February 7, 1947. In 1966, Allwine started work in the mailing room at the Disney studios, before working in the sound effects department under Jimmy MacDonald.  After auditioning for the role, Allwine became the voice of Mickey Mouse from 1977 until his death in 2009. He succeeded MacDonald, who in 1947 had taken over from Walt Disney himself, who had performed the role since 1928 as well as supplying Mickey’s voice for animated portions of the original The Mickey Mouse Club television show (ABC-TV, 1955–59).

Allwine’s first appearance as Mickey was voicing the animated lead-ins for The New Mickey Mouse Club in 1977. His first appearance as Mickey for a theatrical release was in the 1983 featurette Mickey’s Christmas Carol. In the same film, he voiced a Santa Claus on the street appealing for charity donations at the start of the movie, Moley (who appears with Ratty) “collecting for the poor”, and one of the two weasel undertakers in the Christmas future scene.

He also starred in films such as The Great Mouse Detective (1986), Who Framed Roger Rabbit (1988), The Prince and the Pauper (1990) and Mickey, Donald, Goofy: The Three Musketeers (2004), and the TV series Mickey Mouse Works (1999-2000), Disney’s House of Mouse (2001-2003), and Mickey Mouse Clubhouse (2006-2009). He has provided Mickey’s voice for nearly every entry in the popular Kingdom Hearts series of video games, which was done in collaboration with Japanese video game company Square Enix.  In addition to his voice work, Allwine had also been a sound effects editor on Disney films and TV shows including Splash (1984) and Three Men and a Baby (1987); as well as Innerspace (1987), Alien Nation (1988) and Star Trek V: The Final Frontier for other studios.  He played rhythm guitar for the band Davie Allan & the Arrows, including on the 1967 single Cycledelic.

In 1991, Allwine married Russi Taylor, who has voiced Minnie Mouse since 1986 and were named Disney Legends in 2008. Allwine died of acute diabetes on May 18, 2009, he was survived by his children from previous marriages: Erin, Peter, Christopher, and Joshua, and a grandson named Isaac. His final performance was in the Mickey Mouse Clubhouse episode “Choo Choo Express” and the English-language version of Kingdom Hearts 358/2 Days, which are dedicated to his memory. In 2013, his voice was also used in the English-language version of Kingdom Hearts HD 1.5 Remix.