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An extraction and purification method for soluble polyphenol oxidase (sPPO) and membrane-bound polyphenol oxidases (mPPO) from Agaricus bisporus was developed involving temperature-induced phase partitioning, fractional precipitation with ammonium sulfate, and DEAE anion exchange chromatography. The purity, molecular mass and enzymatic reaction kinetics of purified PPO were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE and Michaelis-Menten equation. The specific activities of sPPO and mPPO toward catechol were 6 912.88 and 19 092.94 U/mg, and increased by 12.20 and 10.86 times, respectively, compared with that of crude PPO. The results of enzymatic reaction kinetics showed that the catalytic activities of sPPO and mPPO toward different substrates were greatly different and were higher toward catechol. mPPO had a stronger affinity to the substrate catechol and a higher reaction rate than sPPO. The optimum pH and temperature for both PPO enzymes were 6.8 and 30 ℃, respectively. This study may lay a foundation for future research on mass spectrometry identification and enzymatic properties two forms of PPOs from A. bisporu and inhibition of enzymatic browning caused by PPO.