The Belgian, who won seven grand slam events in a glittering career, made the announcement in her homeland on Wednesday.
The top seed suffered a third-round loss to Dinara Safina at the Qatar Telecom German Open in Berlin last week and withdrew from this week's Internazionali BNL d'Italia due to fatigue.
"I'm at the end of the road," Henin, winner of 41 WTA singles titles, announced at a news conference on Wednesday. "I have experienced everything I could have. I have lived completely for tennis.
"I am relieved and proud of what I achieved."
She insists her decision is final and she will not make a U-turn at any point in the future.
"A new future is ahead and I won't go back on this decision," she added.
Henin has previously admitted she would like to have more time to study, travel and generally escape from the pressures of top-level tennis.
Unable to physically match up with many of the players on tour at just 5ft 6in, Henin utilised her tremendous speed and coverage to go with her trademark shot, a devastating one-handed backhand, to vault to the top of the women's game.
She has topped the WTA rankings for more than a year - since March 2007 - with Wimbledon the only major title to have eluded her.