Olyphant was born in Honolulu, Hawaii to John Vernon Bevan and Katherine Olyphant. His father was a college teacher who worked for a winery. He is a descendent of Cornelius Vanderbilt. Olyphant was raised in Modesto, California, and began competitive swimming at the age of 6. At high school, Olyphant started training seriously under swim coach Darrell Lohrke, who came to Modesto via Arden Hills Swimming and Tennis Club. Olyphant became a nationally ranked swimmer. He was a finalist at the USA Swimming Short Course National Championships in 1986 in the 200 Individual Medley. He graduated from Modesto's Fred C. Beyer High School that year.
Olyphant then decided to move to New York City to study acting rather than pursue a master's degree in fine arts. He took acting classes with William Esper at William Esper Studio for two years, and then started looking for acting work. Olyphant made his professional off-Broadway debut in 1995 in the Playwrights Horizons' production of The Monogamist in the role of Tim Hapgood. He received the Theatre World Award for Outstanding Debut Performance for his performance. He subsequently appeared in the one-man play The SantaLand Diaries in 1996, which was written by David Sedaris and adapted and directed by Joe Mantello, and in the play Plunge.
Olyphant returned to Los Angeles and found work in television. In 1995 he landed his first TV role in a pilot for a remake of the series 77 Sunset Strip, produced by Clint Eastwood. He was cast in the pilot of Mr. & Mrs. Smith (1996), a short-lived CBS spy series, as Scooby, Mr. Smith's (Scott Bakula's) assistant. Olyphant appeared in 3 episodes of the acclaimed police drama High Incident and in the 1997 Hallmark Channel TV film Ellen Foster. A role in the HBO movie When Trumpets Fade was next in 1998, which was a dramatization of the 1944 Battle of Hürtgen Forest. This was followed by a guest starring role in the Sex and the City episode "Valley of the Twenty Something Guys" as one of Carrie Bradshaw's boyfriends.
Olyphant made his feature film debut with a small part in The First Wives Club (1996). He appeared briefly in A Life Less Ordinary (1997), which starred Ewan McGregor, but gained notice appearing in the horror film Scream 2 (1997), as a cynical film student, Mickey. He portrayed the drug dealer Todd Gaines in the comedy-drama Go (1999), directed by Doug Liman, which also starred Katie Holmes, Scott Wolf and Sarah Polley, and featured a script by first time screenwriter John August. His next film roles were in Advice from a Caterpillar (1999), No Vacancy (1999), The Broken Hearts Club: A Romantic Comedy (2000) and Gone in 60 Seconds (2000).
Olyphant played the lead role of Sheriff Seth Bullock in HBO's Deadwood for three seasons from 2004 until 2006. Olyphant considered it to be one of his first lead roles as a "complicated" and "complex" character.
Olyphant served as the daily on-air, unpaid sports commentator for Joe Escalante's morning show on the LA radio station Indie 103.1, until the channel's demise in January 2009.
In the spring of 2010, Olyphant landed the lead role of Raylan Givens in the FX series Justified. His character is a 21st-century U.S. Marshal who is banished back to his home state of Kentucky for questionable actions in Miami. Givens wears a Stetson hat and behaves like an Old West sheriff. The show is based on a character created by Elmore Leonard in the novels Pronto and Riding the Rap, and the short story "Fire in the Hole," which served as the basis for the pilot. Olyphant lives in Westwood, Los Angeles with his wife and their three children. His first child, daughter Grace Katherine was born in 1999 followed by son Henry and daughter Vivian. The couple were married in July 1991.