Abstract: Two kind of extracellular β-glucosidases were purified to homogeneity from an Aspergillus niger by ammonium sulfate precipitation, Phenyl Sepharose CL-4B hydrophobic interaction chromatography, DEAE-Sepharose ion exchange chromatography and Sephacryl S-200 HR gel layer chromatography. One of them was a highly glucose-tolerant novel β-glucosidase with a Ki of 41.01 mmol/L, and the final purification factor 56.7 times and 22.66% yield were obtained. The enzyme single subunit mole-cular weight was about 114.6 ku which was identified by SDS-PAGE. The Km and vmax values of the enzyme were 0.904 mmol/L and 1.08 μmol/min, respectively, using p-nitrophenyl-β-D-glucopyranoside(pNPG) as a substrate. The optimum reaction temperature and pH value for β-glucosidase were 60℃ and pH value 4.0, respectively. The enzyme was stable in the pH value range of 3.0-7.0 and up to 60℃. The enzyme was greatly inhibited by Ag+. Other metal ions tested and EDTA hand no effects on the activity of β-glucosidase. Different organic solvent had different effects on the activity of β-glucosidase, methanol and 1-butanol enhanced the activity, whereas acetone and acetonitrile inhibited the activity obviously.