AbstractMedium components for β-glucosidase (BGL) production in Schizophyllum commune KUC9397 were optimized using a central composite design and response surface methodology. From the various medium components tested, cellulose, soy peptone, and thiamine HCl were selected as the optimal carbon, nitrogen, and vitamin sources, respectively. The highest BGL production was obtained with 2.96% cellulose, 2.30% soy peptone, and 0.11% thiamine HCl. BGL production in the optimized medium was increased 7.2-fold compared to production in an unoptimized medium. Crude enzyme preparation from S. commune KUC9397 was used to saccharify pretreated lignocellulosic biomass. The crude enzyme preparations showed statistically equal saccharification rates as Cellobiase, a commercial BGL. This finding indicates that crude enzymes produced by S. commune KUC9397 have good potential for application in cellulosic biomass conversion systems in place of Cellobiase.