A two-step optimization procedure using central composite design with four factors (concentrations of maltrin and corn steep liquor (CSL), agitation speed and inoculation ratio) was used in order to investigate the effect of these parameters on the polygalacturonase (PG) enzyme activity, mycelia growth (biomass) and morphology (pellet size) of Aspergillus sojae ATCC 20235. According to the results of response surface methodology (RSM), initial concentrations of maltrin and CSL and agitation speed were significant (p < 0.05) on both PG enzyme production and biomass formation. As a result of this optimization, maximum PG activity (13.5 U/ml) was achievable at high maltrin (120 g/l), at low CSL (0 g/l), high agitation speed (350 rpm) and high inoculation ratio (2 × 107 total spore). Similarly, maximum biomass (26 g/l) could be obtained under the same conditions with only the difference for higher level of CSL requirement. The diameter of pellets in all optimization experiments ranged between 0.05 and 0.76 cm. The second optimization step improved the PG activity by 74% and the biomass by 40%.