The aim of this study was to determine the activity of β-galactosidase in the crude extracts of Pleurotus ostreatus in the presence and absence of various heavy metals. β-galactosidase (EC 3.2.1.23), is a hydrolase enzyme which helps in the hydrolysis of lactose into monosaccharides. Characterization of β-galactosidase from Pleurotus ostreatus was achieved using the substrate 2-nitrophenyl β-D-galactopyranoside (ONPG). The pH and temperature profiles of β-galactosidase showed maximum activity at pH 3.0 and at 50°C, respectively. The Vmax and Km values of β-galactosidase using ONPG as a substrate was found to be 0.571 μmol/min and 0.307 mM, respectively. These results revealed that the β-galactosidase activity in the crude extracts of Pleurotus ostreatus was changed in the presence of different heavy metals. The results indicated that Hg2+ and Mo2+ have an uncompetitive inhibition on the β- galactosidase activity in the extract of Pleurotus ostreatus by decreasing both Km and Vmax values. while Al3+, Cu2+, Cr3+, Zn2+ and Ni2+ showed mixed inhibition activity by decreasing Vmax values and by increasing Km values. However, Pb2+ was found to act as a non-competitive inhibition by decreasing Vmax value. The findings suggested that crude extract of Pleurotus ostreatus can be used as a source of β-galactosidase for medical and industrial purposes.