Abstract: Response surface analysis method was applied to optimize the ultrasonic-assisted extraction process of Pleurotus nebrodensis polysaccharides ( PNPs) , and evaluate the antioxidant activities in vitro of PNPs.On the basis of single-factor test, the ultrasonic treating time, extraction temperature and the ratio of water volume to raw material weight ( W/M ratio) were analyzed and optimized by the Box-Behnken design.The optimized parameters of the ultrasonic-assisted extraction technology were obtained as follows: extraction temperature was 56 ℃, ultrasonic treating time was 45 min, W/M ratio was 41∶ 1, extraction frequency was 2 times, and the extraction rate of the PNPs was 12.26%.Compared with the hot water extraction method which the extraction temperature was 77 ℃, extraction time was 189.6 min, W/M ratio was 46∶ 1, extraction frequency was 2 times, the extraction rate of the polysaccharides was increased by 44.41%. Antioxidant activities in vitro of the PNPs indicated that the hydroxyl racials and DPPH racials scavenging rates of PNPs were 98.39% and 60.8% respectively when its concentration was at 4 mg/m L.