Gattuso was due to start his role as the new man in charge at the Stadio Artemio Franchi dugout at the start of next month but differences with the club’s board regarding transfer targets has led to both parties severing ties.
Gattuso left Napoli at the end of the 2020/2021 season after the Partenopei failed to qualify for next season’s UEFA Champions League and was appointed as Fiorentina boss barely 48 hours later.
The 43-year-old leaves Fiorentina without taking charge of a single game.
A statement on the club's official website reads: "ACF Fiorentina and Mister Rino Gattuso, by mutual agreement, have decided not to follow up on the previous agreements and therefore not to start the next football season together.
"The Company immediately set to work to identify a technical choice that will guide the Viola team towards the results that Fiorentina and the City of Florence deserve."
Fiorentina made three managerial alterations in the course of last season and finished 13th on the Serie A table.
Some of the early frontrunners tipped to replace Gattuso include former Leicester City and Chelsea manager Claudio Ranieri and ex Juventus boss Andrea Pirlo.