"I'm announcing my definitive retirement from football and I thank everyone who has helped make my career so successful," said the former Fiorentina and AS Roma striker in a statement sent to Argentine media from Qatar on Sunday.
The decision came two days after Batistuta ended his contract with Qatari club Al Arabi following an injury-plagued year in which he played only three matches and failed to score.
Batistuta, who hit 56 goals in 78 internationals, said he was planning to coach in the future.
"I will certainly continue in football in a different role," he added. "I will continue with my contractual, publicity and managerial commitments and finish my studies for coaching."
Batistuta played in the 1994, 1998 and 2002 World Cup finals, his last international goal coming in the 1-0 win over Nigeria in the 2002
His Argentina career also produced frustration when he was overlooked for long periods by former coach Marcelo Bielsa, who often preferred Hernan Crespo in attack and would not field the pair together because he said their styles were incompatible.
Batistuta made his professional debut with Newell's Old Boys in 1989. He also spent six months with River Plate before moving to Boca Juniors, becoming one of the few players to play for the two bitter Buenos Aires rivals.
He joined Italy's Fiorentina in 1991 and spent nine seasons there, including one in the second division after he preferred to help the club back to the top flight rather than move elsewhere.
Batistuta joined AS Roma in 2000 and had a brief spell with Inter Milan in 2003 before leaving for Al Arabi.
