Abstract: The βglucosidase were separated and purified from cellulase solutions produced by Trichoderma reesei, Aspergillus niger, and mixed enzymes of T. reesei and A. niger through (NH4)2SO4 fractional precipitation, HiPrep 26/10 gel filtration, Source 15 Q and source 15 S ionexchange columns, and HiTrep 16/60 Sephacryl S200 HR gel column. Both β1,4glucosidase showed apparent homogeneity in SDSPAGE. The estimated molecular masses of β1,4glucosidase were 68 and 129 ku from T.reesei and A.niger, respectively. Two kinds of βglucosidases were purified from mixed culture of T.reesei and A.niger, two single subunit molecular masses were about 134 ku and 66.2 ku which are similar to the single culture. Three sources of βGA through multistep purification the purification multiple were 37.25, 40.21, 30.12 respectively. The recovery of enzyme activity were 20.12 %,23.21 %,28.56 %.