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).