The 36-year-old joined La Viola in July 2013 after bringing an end to his 18-year career at AC Milan, and made 26 appearances for them in all competitions.
He was keen to stay at the Florence outfit but there was no contract offer on the table, and so Ambrosini has reluctantly resigned himself to moving on.
He told reporters: "My adventure with Fiorentina ends here. I thank Florence, it has been a fantastic year.
"I have never hid I would have liked to continue playing here, but the club told me that they have made their decision.
"I wish my teammates well. I remain tied to this club and to president Andrea Della Valle who always allowed me to feel at home."
Ambrosini won 35 caps for the Italian national side between 1999 and 2008.