Abstract: In this study,three genes( O158,AO and O76) encoding β-galactosidases from Aspergillus oryzae were cloned and expressed.The recombinants GS115( p PIC-O158),GS115( p PIC-AO) and GS115( p PIC-O76) were constructed and the recombinant enzymes were subsequently obtained. Further analysis showed that the p H optima of O158,AO and O76 were 4.0,5.5 and 7.0,respectively,and their optimal temperatures were all 50 ℃.These recombinant enzymes were stable in the p H range of 5.0 ~ 7.5 or at temperatures ranging from 30 to 40 ℃. Their activities were enhanced by Mn2 +,but significantly inhibited by Fe2 +,Cu2 +and Zn2 +. O158,AO and O76 only exhibited hydrolytic and transgalactosyl activities toward lactose.The affinity and catalytic efficiency of AO towards lactose were higher than those of O158 and O76. Besides,O42 was not existed in the test strain although it was predicted in the reference genome sequence. The heterologous expression and biochemical properties of threeβ-galactosidases from A.oryzae were comprehensively investigated in this study,which lays a solid foundation for their large-scale productions and industrial applications.