In order to maximize the riboflavin production by a mutant strain Ashbya gosspyii, the optimization of medium and fed-batch fermentation were performed. As carbon sources, glucose and soybean oil were necessary for the riboflavin overproduction. Optimal concentrations of glucose and soybean oil in the flask cultures were found to be 3.0% and 0.5%, respectively, in a complex medium containing corn steep liquor(CLS) 1%. Among the various organic nitrogen sources tested, CSL was the most effective one both for the cell growth and riboflavin overproduction.