The Italy international had been strongly linked with a move to Juventus this month.
However, Fiorentina have confirmed they will not sell the player, who is under contract with La Viola until June 2013.
A statement from the club read: "Seeing the continuous references in the press regarding prospective deals on the transfer market in relation to Alberto Gilardino, Fiorentina would like to communicate the player is absolutely not for sale.
"The club intends to focus on the objective of growing and investing to improve the actual squad."
Gilardino, 28, is Fiorentina's top scorer with five goals his season.
His side head into the encounter at the Renato Dall' Ara stadium looking to spark their season into life.
Sinisa Mihajlovic's men have won just one of their last four games and a 2-1 defeat at Udinese before the winter break was their seventh loss of the campaign.
They lie 15th in the standings.
"We know we have not done well so far but now we hope that 2011 will begin on the right foot, starting with the game at Bologna," Fiorentina midfielder Juan Vargas said.
The Viola have not won on the road since last March and face a Bologna side that has looked solid at home so far.
Alberto Malesani's men have lost just once in Bologna this season, a 3-0 defeat at the hands of Serie A leaders AC Milan last month.
Bologna lie one point clear of Fiorentina heading into the match.
Malesani will have all of his players available with striker Marco Di Vaio having recovered from a foot injury.
The visitors will be without suspended midfielder Marco Donadel and injured trio Mario Santana, Stevan Jovetic and Sebastien Frey.