Lajoie was born in Longueuil, Quebec, Canada, and raised in the South Shore of Montreal, Quebec. His father is Québécois and his mother is English-Canadian, and he is the third of nine children. He graduated from Dawson College's professional theatre program in 2002.
Since early 2003, Lajoie has portrayed an English-Canadian musician named Thomas Edison in Radio-Canada's French-Canadian sitcom L'Auberge du chien noir. Lajoie began his career as a comedy musician in 2006. His performances include music, skits, and some stand-up comedy. Jon Lajoie has also released 3 studio albums, the first two featuring comedy music. His third album took a folk music direction independent of comedy and was released under the moniker 'Wolfie's Just Fine'.
Lajoie filmed an episode of Comedy Central Presents on November 7, 2009. It premiered on March 5, 2010. The same special aired uncensored as part of Comedy Central's Secret Stash on June 6 featuring the world premiere of the video for his song "Pop Song".
Lajoie was featured in the 2014 movie Let's Be Cops, as the boss of Damon Wayans, Jr.'s character. He was also featured in Quentin Dupieux's 2013 movie Wrong Cops as Officer Regan.