Abstract: In this experiment, the main research material was dried selenium-enriched Pleurotus ostreatus, in order to get optimization of extraction technology for water soluble protein and determine its antioxidant activity in vitro, single factors and orthogonal experiments were designed. The results showed that the best conditions for extraction were described as follows:extraction temperature 30 ℃, extraction time 4 h and the ration of raw material to water 1∶ 30, under this conditions, the content of water soluble protein reached ( 8.5 ± 0.29) mg/mL.The water soluble protein had the capacity of scavenging superoxide anion free radical, hydroxyl radical and ABTS radical, and the scavenging rate could get higher than 50% at the concentration of 1.0 mg/mL.