A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer(ITS) rDNA gene sequence.The present study is to ferment,purify and characterize a β-glucosidase from T.cylindrosporum gams.The enzyme was purified to homogeneity by sulfate precipitataion,diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and a recovery of 12.27%.The molecular weight(Mw) of the β-glucosidase was estimated to be 58600 by SDS-PAGE,which is much lower than that of the enzyme from other fungi.The β-glucosidase shows high activity over a wide range of temperature from 35℃ to 70℃ and the optimum pH is approximately 2.4.Then the kinetic parameters Km(0.85 mmol/L) and vmax[85.23 mmol/(L·s)] of the β-glucosidase were studied,respectively with high affinity p-nitrophenyl-β-D-glucopyranoside(p-NPG) as the substrate,inhibition constant Ki(39.5 mmol/L) with the tolerance of glucose.Although β-glucosidases have been purified and characterized from several other sources,T.cylindrosporum β-glucosidase with the unique optimal pH and higher tolerance of glucose distinguished from others makes the β-glucosidase a potential application in simultaneous saccharification and fermentation(SSF). A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. The present study is to ferment, purify and characterize a β-glucosidase from T. cylindrosporum gams The enzyme was purified to homogeneity by sulfate precipitataion, diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and a recovery of 12.27%. The molecular weight (Mw) of the β- glucosidase was estimated to be 58600 by SDS-PAGE, which is much lower than that of the enzyme from other fungi. The β-glucosidase shows high activity over a wide range of temperatures from 35 ° C to 70 ° C and the optimum pH is approximately 2.4. Then the kinetic parameters Km (0.85 mmol / L) and vmax [85.23 mmol / (L · s)] of the β-glucosidase were studied, respectively with high affinity p-nitrophenyl- β- D-glucopyranoside (p- NPG) as the substrate, inhibiti on constant Ki (39.5 mmol / L) with the tolerance of glucose. Although β-glucosidases have been purified and characterized from several other sources, T.cylindrosporum β-glucosidase with the unique optimal pH and higher tolerance of glucose distinguished from others makes the β-glucosidase a potential application in simultaneous saccharification and fermentation (SSF).