The Jamaican world record holder has already announced appearances in the 200 at Oslo's Bislett Games on June 13 and at the Paris Golden League meeting on July 6 ahead of the world championships in Moscow in August.
Bolt, who became the only man to win the 100-200 metres double at successive Olympics during last year's London Olympics, is also due to compete in a 150 metres race on Brazil's Copacabana beach on March 31.
The Golden Gala appearance will be Bolt's third in a row at Rome's Olympic stadium. He set a time of 9.91 seconds in 2011 and 9.76 last year in front of more than 50,000 spectators.
Rome is the fifth round of the 14-event Diamond League calendar.
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