AbstractExtracellular fungal cellulases are key enzymes for the degradation of lignocellulosic biomass. Greater production of these enzymes could reduce the cost of biofuels production. In this study, the basal medium for cellulase production by a Penicillium janthinellum mutant (EU2D-21) in submerged fermentation conditions was optimized using response surface methodology (RSM). Initial studies using a Plackett-Burman design (PBD) showed that (NH4)2SO4 and urea are significant factors for improving β-glucosidase and FPase production. A central composite design (CCD) was applied to obtain the maximum response, which resulted in the optimal production of β-glucosidase (5.79 IU/mL) and FPase (5.76 IU/mL). These values were 1.87 and 1.67 times higher than the corresponding values obtained under un-optimized conditions.