Abstract: Pleurotus ostreatus is a white-rot fungus secreting different enzymes involved in lignin and cellulose degradation. Laccase and isoenzymes activities of P. ostreatus (ACCC 52857) were tested in the presence of different concentrations of Cu²⁺, Pb²⁺ or Cd²⁺ under shaking conditions. The mycelia growth and laccase activities were assayed, and laccase isoenzymes were analyzed using non-denaturing polyacrylamide gel electrophoresis. The results showed that the laccase activity increased with treatments of Cu²⁺, Pb²⁺ or Cd²⁺ in the medium compared with the control group. The highest laccase activity (565.6±5.2) nkat/mL was obtained on the 9^th day of cultivation in the control group, while laccase activity continued to increase in the presences of Cu²⁺, Pb²⁺ or Cd²⁺. The highest laccase activities -(8612.1±840.0), (2204.0±347.0) and (2928.0±217.7) nkat/mL-were obtained on the 20^th, 15^th and 17^th day of cultivation by adding 3000 μmol/L Cu²⁺, 2000 μmol/L Pb²⁺ or 150 μmol/L Cd²⁺ to the medium, respectively. The results of non-denaturing polyacrylamide gel electrophoresis indicated that the two most abundant isoenzymes (Lacc2 and Lacc10) in liquid culture showed different expression patterns in response to the concentration variations of Cu²⁺, Pb²⁺ and Cd²⁺. Lacc10 activity was increased greatly with high concentration of Cu²⁺ in the medium, while Lacc2 activity was increased by Pb²⁺. In the presence of Cd²⁺, Lacc2 activity was slightly suppressed, while Lacc10 showed a reverse trend. The results may shed light on the biological functions and the regulation mechanisms of laccase in P. ostreatus.