After graduating from the London Academy of Music and Dramatic Art in 2006, he played a series lead in the Innocence Project for BBC television. He also appeared as a 14-year-old Theo in the hard-hitting Channel 4 drama Clapham Junction.

He made his stage debut in Saint Joan at the Royal National Theatre, followed by playing Albert in the original production of War Horse at the same venue. In 2008 he appeared in Piranha Heights, a new play by Philip Ridley, at the Soho Theatre, and in Cradle Me, a new play by Simon Vinnicombe, at the Finborough Theatre. He also played Albert in John Tams' adaptation of War Horse for BBC Radio 2 on 8 November 2008.

In 2009, he appeared in the play Over There at the Royal Court Theatre alongside his twin brother from 25 February to 21 March. He spent November 2009 in Romania filming The Whistleblower with Rachel Weisz, Vanessa Redgrave and Monica Bellucci.

He played Prokopion in the 2010 film Clash of the Titans. He plays Lee in Philip Ridley's Heartless, also starring Jim Sturgess and Noel Clarke, released in May 2010. He appeared in Killing Bono, starring Ben Barnes and Pete Postlethwaite. He also had a leading role in the independent Scottish film You Instead, which was filmed at T in the Park 2010.

In 2011, he played the eponymous hero of The History of Titus Groan, a cycle of six BBC Radio 4 dramas based on the books of Mervyn Peake dramatised by Brian Sibley. He also filmed Late Bloomers with william Hurt and Isabella Rossellini. He also starred in Attack the Block, a Film Four production, alongside John Boyega, Nick Frost and Jodie Whittaker. He also directed a music video for girl band Boxettes, whose lead singer Bellatrix comes from Sandford.

In 2013 Treadaway lived in a flat in North London, with his girlfriend Ruta Gedmintas, his twin brother Harry, and a friend who played in a band.

In April 2018, Luke played the part of Dr Arthur Calgary in a BBC One three-part adaptation of the Agatha Christie novel Ordeal by Innocence.