The veteran midfielder made 21 league appearances for the Stadio Artemio Franchi outfit after calling time on his 18-year Milan career at the end of the 2012-13 campaign.
However, in a press conference on Tuesday, Ambrosini confirmed that he would not be spending a second season in Florence.
"I'm here today to announce that my time with Fiorentina has come to an end," he said. "I want to thank the city of Florence. It's been an exceptional, wonderful year and I've met some amazing people.
"I wish my team-mates all the very best of luck for next season."
And the 36-year-old believes his future may now lie outside his homeland.
"I want to give my future some careful thought," he continued. "Unless something totally unexpected happens I think I've probably finished with Italian football."
Ambrosini's agent claimed he was keen on new contract last week, but the player himself hinted an extension had never been discussed, adding: "You only talk about money when you plan to go on together."
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