Harris was born in Tunica, Mississippi. After publishing two stand-alone mysteries, Harris began the lighthearted Aurora Teagarden books with Real Murders, a Best Novel 1990 nomination for the Agatha Awards. Harris wrote several books in the series before the mid-1990s when she began branching out into other works. She did not resume the series until 1999, with the exception of one short story in a Murder, She Wrote anthology titled "Murder, They Wrote".

In 1996, she released the first in the Shakespeare series featuring cleaning lady detective Lily Bard, set in rural Arkansas. Harris "lives in small-town Arkansas", according to a New York Times interview. The fifth book in the series, Shakespeare's Counselor, was printed in fall 2001, followed by the short story "Dead Giveaway" published in the Ellery Queen Mystery Magazine in December of the same year. Harris has stated on her website that she has finished with the series.

After Shakespeare, Harris created The Southern Vampire Mysteries series about a telepathic waitress named Sookie Stackhouse who works in a northern Louisiana bar. The first book in the series, Dead Until Dark, won the Anthony Award for Best Paperback Mystery in 2001. Each book follows Sookie as she tries to solve mysteries involving vampires, werewolves, and other supernatural creatures. The series has been released in Australia, New Zealand, Japan, Spain, Greece, Germany, the Netherlands, Belgium, France, Italy, Argentina, Poland, Serbia, Brazil, Great Britain, Ireland, Mexico, Norway, Finland, Sweden, Denmark, Lithuania, Hungary, Bulgaria, Portugal, Iceland, Czech Republic, Romania, Estonia and Israel. Harris wrote thirteen novels in the series. The thirteenth and final novel in the series, Dead Ever After, was published in May 2013, with a supplemental book, After Dead, released in October 2013.

October 2005 marked the debut of Harris's new series entitled The Harper Connelly Mysteries, with the release of Grave Sight. The series is told by a young woman named Harper Connelly, who after being struck by lightning, is able to locate dead bodies and to see their last moments through the eyes of the deceased. In October 2010, it was announced Harper Connelly's series had been optioned for a television series named Grave Sight.

Sookie Stackhouse had proven to be so popular that Alan Ball, creator of the HBO television series Six Feet Under, undertook the production of a series based upon The Southern Vampire Mysteries. He wrote and directed the pilot episode for the series, True Blood, which premiered on September 7, 2008 on HBO. The television show was a critical and financial success for HBO running 7 seasons through the 2014 year.

2014 marked the debut of the Cemetery Girl series, a graphic novel series co-written with Christopher Golden and illustrated by Don Kramer.

The second show to be based on Charlaine Harris's work is Midnight, Texas. The show premiered on NBC in 2017.