Cellobiase production was carried out by recombinant Pichia pastoris containing Aspergillus niger cellobiase gene,and the main fermentation parameters were optimized.The fermentation result in shaking flasks showed that the optimal concentrations of glycerol and corn steep powder were 2.0%(w/v) and 3.0%(w/v) respectively,and the optimal pH value was 6.0.Methanol as carbon source and inducer has obvious effect on cellobiase production by recombinant Pichia pastoris.The activity of cellobiase could reach 9.01 IU/mL after 8d fermentation by addition of 1.0%(v/v) methanol,0.2%(w/v) glycerol and 0.3%(w/v) corn steep powder every 24 hours.The result is valuable in improving the efficiency of cellulose hydrolysis by cellulase and promoting industrialization of ethanol production from cellulosic material.