The optimal extraction conditions of polypeptide from Pleurotus eryngii mycelium was ivestigated, and activity of the antioxidant was evaluated in this study. Based on the single-factor tests, the extraction conditions of polypeptide from P. eryngii mycelium were optimized by Response Surface Methodology(RSM) to increase the extraction rate of polypeptide. Moreover, the polypeptide was purified with Sephadex-50, and the antioxidant activity of polypeptide was assessed using several assay model systems in vitro. The optimal extraction conditions of polypeptide were pH 8.85, extraction time 4.17 h, liquid-to-material ratio 111.86 (g/mL). Under the optimal extraction conditions, the maximum extraction rate of polypeptide was 50.88%. Extracted polypeptide showed good antioxidant activity in a dose-dependent manner.