He made his screen debut on the "Black Friday" episode of the TV series Medic (airdate November 21, 1955), the first of more than 200 episodic roles by mid-2000. His first movie appearance was as an uncredited extra in The Ten Commandments (1956), playing a golden calf idolator and also visible in a scene in a chariot behind that of Yul Brynner. Vaughn's first credited movie role came the following year in the Western Hell's Crossroads (1957), in which he played the real-life Bob Ford, the killer of outlaw Jesse James.

Vaughn's first notable appearance was in The Young Philadelphians (1959) for which he was nominated for a Supporting Actor Academy Award. Next he appeared as gunman Lee in The Magnificent Seven (1960), a role he essentially reprised 20 years later in Battle Beyond the Stars (1980), both films being adaptations of filmmaker Akira Kurosawa's 1954 Japanese samurai epic, Seven Samurai. Vaughn played a different role, Judge Oren Travis, on the 1998-2000 syndicated TV series The Magnificent Seven. Vaughn is the only surviving member of the title cast of the orginal 1960 film.

From 1964-1968, he starred as "Napoleon Solo", the eponymous man from U.N.C.L.E. ("United Network Command for Law and Enforcement"), along with British co-star David McCallum. Following the end of that hit series — which had spawned a spin-off show, large amounts of merchandising, and overseas theatrical movies of reedited episodes — Vaughn continued to act in television and in mostly B movies. He starred in two seasons of the popular Gerry Anderson detective series The Protectors in the early 1970s, and a decade later co-starred with George Peppard in the final season of The A-Team.

In 2004, after a string of guest starring roles on series such as Law & Order, in which he had a recurring role during season eight, Vaughn experienced a career resurgence when he began co-starring in the BBC series Hustle, made for the UK's BBC One, also broadcast in the United States on the cable network AMC. In the series Vaughn plays elder-statesman con artist Albert Stroller, a father figure to a group of younger grifters. In September 2006 he guest starred in Law & Order: Special Victims Unit.

Vaughn married actress Linda Staab in 1974. They have adopted two children, Cassidy (b. 1975) and Caitlin (b. 1981). They also have a Labrador Retriever mix named Sam (named after the beer, Sam Adams), which was adopted after the death of their previous dog, a Bichon Frise named Peaches.

Politically, he is a moderate Democrat.