The drama stars Jordana Spiro as Grace Devlin, a medic who must work off her brother's debt to the mob.
Reservoir Dogs star Madsen will play Russell King, a master criminal who turns to Devlin when he requires emergency surgery, TV Guide reports.
The 55-year-old actor is best known for his film credits, including roles in Free Willy, Donnie Brasco, Kill Bill and Sin City.
Also joining The Mob Doctor is Jennifer Beals (Flashdance, The Chicago Code), who will appear in at least three episodes as Celeste LaPree, a former high-class call girl who used to date Constantine Alexander prior to his stint in prison.
"We wrote the role of Celeste LaPree specifically with [Beals] in mind," said show creator Josh Berman. "In a world of organised crime, dominated by men, Celeste LaPree has made a name for herself."
The Mob Doctor has struggled in the ratings, attracting 5.1m viewers to Fox on its September 17 debut, but pulling in just 3.4m for its most recent episode.
The series is currently on a month's hiatus and will return to Fox with new episodes on Monday, November 5.
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Watch a trailer for The Mob Doctor below: