The bottleneck of producing bioethanol from lignocellulosic materials is cost. This study adopted response surface methodology to optimize the cellulase production by Penicillium sp. CLF-S. Three variables of carbon nitrogen ratio, liquid-solid ratio, and buffer solution volume that have significant effects on the cellulase production were determined. After analyzing the results of full factorial central composite design, a quadratic polynomial model was established. The final optimal values of the three variables were as follows: carbon nitrogen ratio was 14.65 (mol:mol), liquid solid ratio was 34.36 (ml:g), and buffer solution was 13.29 ml per 100 ml media. The maximal cellulase activity could be up to 1,215.38 u/ml, which was 84.64% higher than the control value. The optimized coarse enzyme solution was extracted out of the cultivation system in order to decompose the corn stover, and the concentration of glucose (6.32 g/l) was 2.47 times that of hydrolysis with non-optimized cellulase.