Lucas's association with Vic Reeves and Bob Mortimer began in 1992. He appeared in The Smell of Reeves and Mortimer in 1995, and went on to star with them in Shooting Stars. He quickly rose to fame as George Dawes, a giant baby (often dressed in an adult-sized romper suit) who would deliver a string of meaningless gags and insults before delivering the teams' scores, while sitting at and playing a drum kit. He also appeared on occasion as Marjorie Dawes, George's mother, who also appears in Little Britain.

He again appeared with Reeves & Mortimer in the BBC TV series Randall & Hopkirk (Deceased) and Catterick, in a variety of roles. In 1999, Lucas paired with David Walliams, with whom he had already worked in Mash and Peas and Sir Bernard's Stately Homes, to create Rock Profile, a comedy show spoofing famous musical personalities. It is notable for being one of their first comedic collaborations.

His music video appearances include the Damien Hirst-directed video for Blur's "Country House", "Jesusland" by Ben Folds in 2005, "I'm with Stupid" by Pet Shop Boys, and "Vindaloo" by Fat Les.

Lucas ventured into stage musicals in 2002, when he took a role in Boy George's musical Taboo at The Venue in London. He played the performance artist Leigh Bowery.
Little Britain is Lucas' most commercially successful work. The show came about when trainee BBC radio producer Ashley Blaker, tasked with coming up with ideas for a television or radio series, bumped into Lucas in London. The two were old friends from Haberdashers' and Lucas took Blaker to the Groucho Club, where he told Blaker of his ideas for a sketch show. Little Britain was launched as a radio show on BBC Radio 4 and it later became a TV series.

Among the many characters he plays in the series, which he writes and acts in along with David Walliams, are apparently "disabled" Andy Pipkin, teenage Bristol chav Vicky Pollard, homophobic homosexual Daffyd Thomas, and insensitive slimming club organiser Marjorie Dawes.

In January 2005, Lucas and Walliams were named the most powerful people in TV comedy by Radio Times.
In 2005, he took his first role in a television drama, a supporting part as a Venetian duke in the BBC historical serial Casanova, written by Russell T Davies. Also in 2005, he did voice work in the ITV children's program King Arthur's Disasters alongside Rik Mayall. Since 2006, Lucas has been the voice of the radio and television character Digit Al, devised as part of a public information campaign on digital switchover. On 26 November …

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Lucas is a patron of the Karen Morris Memorial Trust, a UK charity for leukaemia patients and their families. In April 2003, he appeared on Celebrity Who Wants to Be a Millionaire? and won £62,500 for the charity.

Lucas is gay. He first suspected his sexuality at age seven. At a ceremony in Central London in December 2006, he entered into a civil partnership with Kevin McGee. In attendance were Barbara Windsor, Neil Tennant, Elton John, and Courtney Love. They separated and had their civil partnership dissolved through the High Court in 2008.

Lucas was raised as Jewish but has variously described himself as an atheist and a "fairly secular Jew". In 2022, he was the subject of BBC's Who Do You Think You Are? and learned that many of his grandmother's cousins had been murdered in the Holocaust. His grandmother's cousin, Werner Goldschmidt, had lived with the family of diarist Anne Frank in Amsterdam in 1942. In 2021, 83 years after his grandmother left Berlin, Lucas obtained German citizenship.

Lucas is an avid supporter of Arsenal Football Club.

From 2012 to 2015, Lucas lived with his Bridesmaids co-star Rebel Wilson in West Hollywood.

• Boyd Hilton; Matt Lucas; David Walliams (2006). Inside Little Britain. London: Ebury Press. ISBN 978-0-09-191231-4.
• Lucas, Matt (2017). Little Me. London: Canongate. ISBN 978-1786890863.
• Lucas, Matt (2023). The Boy Who Slept Through Christmas. Glasgow: Farshore. ISBN 978-0008519896.