After graduating from RADA in 2000, Mays soon started appearing in a number of supporting roles ranging from a bit part in the BBC soap opera EastEnders in 2000 to playing a pilot in Jerry Bruckheimer's big-budget Pearl Harbor (2001). He was cast in the Mike Leigh film All or Nothing (2002) as Jason, a thug who abused his girlfriend, and also appeared in Leigh's next project, Vera Drake (2004), in which he played Sid, the protagonist's son. His performances for Leigh resulted in further offers of work.

One of Mays's most notable early roles was in the improvised BBC drama Rehab. Directed by Antonia Bird, Rehab was a drama about life inside a drug rehabilitation facility. He starred as Adam, a young heroin addict released from prison and sent directly to rehab. For his performance Mays was awarded the Best Actor award at the Palmare-Reims Television Festival in 2003.

Mays has continued to work regularly, and has appeared in a variety of productions, which have included a part in Johnny Vaughan's sitcom, Top Buzzer (2004); the lead role of Carter Krantz in BBC Three's Funland (2005); as well as film appearances in Atonement (2007), White Girl (2008) and The Bank Job (2008).

Mays and his longtime partner, makeup Artist Louise Burton, have a son, Mylo, and a daughter, Dixie. As of 2005, Mays and his family were living in Crouch End, an area in the London Borough of Haringey. A keen football fan, he is a supporter of Leyton Orient. He is also a fan of British rock band Feeder, having appeared in a 2012 music video.

Projects in 2009 included Hippie Hippie Shake (as '60s alternative figurehead David Widgery, alongside Cillian Murphy and Sienna Miller); a role opposite Anna Friel in the third series of Jimmy McGovern's The Street; a "mark" in the BBC drama serial Hustle; as well as an appearance in the independent British film Shifty, co-starring Riz Ahmed, for which he received a nomination for best supporting actor at the British Independent Film Awards.

Mays starred as Eddie O'Grady in the 2010 film Made in Dagenham. In the same year, he played DCI Jim Keats in the third series of "Ashes to Ashes", in which he portrayed a character that was the antagonist of Philip Glenister's Gene Hunt.

Mays appears in the BBC sci-fi series Outcasts, which started on 7 February 2011, as PAS Officer Cass Cromwell, and in the ninth episode of the 6th series of Doctor Who, titled "Night Terrors," broadcast on BBC One on 3 September 2011.

He had roles in No One Gets Off in This Town and a supporting role in the Steven Spielberg film The Adventures of Tintin: The Secret of the Unicorn. He then played a Criminal on a curfew after serving a 10-year sentence for the murder of his girlfriend when he was 19 in the programme Public Enemies, which aired on BBC One in early January 2012. He played Ronnie Biggs in a 5-part drama called Mrs Biggs.

For much of the latter half of 2013, Mays performed on stage. Performing in Nick Payne's Same Deep Water As Me at the Donmar Warehouse alongside Nigel Lindsay and in the first major revival of Jez Butterworth's debut play, Mojo at the Harold Pinter Theatre. He starred alongside Ben Whishaw, Brendan Coyle, Rupert Grint and Colin Morgan.

From 29 March - 14 May 2016 Mays played the part of Aston in Harold Pinter's play The Caretaker directed by Matthew Warchus at The Old Vic Theatre in London opposite Timothy Spall and George MacKay.

On 11 April 2017 Daniel Mays was nominated for a BAFTA Television Award for Supporting Actor for his role in Line of Duty series 3.