Henin, who lost against Russia’s Svetlana Kuznetsova in the third round of the Australian Open last weekend, revealed that her elbow had been damaged during her exertions in Melbourne.
“In these recent months I have rarely been spared of the pain,” she said.
“The doctors told me my elbow is too fragile and therefore I cannot continue my profession at this high level.”
The 28-year-old Belgian first called time on her glittering career, which saw her amass seven grand slam titles, in 2008 before returning to the WTA tour last January.
Henin reached the final of the Australian Open 12 months ago but her comeback was stunted when she injured a ligament in her elbow during Wimbledon.
“I suffered a lot the last week and every day gave me more and more pain, but I believed that my will would take the upper hand,” she added in an open letter on her personal website.
“I’m in shock, of course. After having considered the advice of doctors, it is now clear and accepted that my career finally ends.”
Henin’s rivalry with fellow Belgian Kim Clijsters dominated the sport in the early part of the millennium as the pair briefly interrupted the dominance of Venus and Serena Williams.
Henin won the US Open in 2003 and 2007, the Australian Open in 2004 and the French Open at Roland Garros four times.