To increase the efficiency of biotransforming saponins in Dioscorea zingiberensis by Trichoderma reesei, four saponin hydrolases (E1, E2, E3 and E4) were purified and characterized. The molecular weights of E1, E2, E3 and E4 determined by SDS-PAGE were 50, 83, 67 and 46 kDa, respectively. E2 and E3 retained high activities at 20-60 °C. Based on HPLC-MS analysis, the substrate preferences of E1, E2, E3 and E4 were carefully studied. Both E2 and E3 could cleave C-26 glycoside, C-3 terminal glycoside, C-3 terminal rhamnoside and C-3 glycoside attached to aglycone in saponins. Four parameters (pH, T, Ca2+ and K+) which influenced the performances of E2 and E3 significantly were optimized for high biotransformation efficiency. After optimization, saponins in Dioscorea zingiberensis were efficiently hydrolyzed by the crude enzyme from Trichoderma reesei, with a diosgenin yield of 94.9%.