The aim of this study was to investigate the effect of ultrasound power at 40 to 80 percent (equal to 232-464 W), extraction temperature (50 to 80 °C) and extraction time (10 to 30 min) on the soluble polysaccharide extraction from the Pleurotus ostreatus. The response surface methodology using Box-Behnken design (with three variables, three levels and 5 replications at central point) was applied to optimize extraction conditions and evaluation of the effects of main factors and their interactions. Antioxidant activity (scavenging ability of OH and DPPH) of extracted polysaccharide were also evaluated. Optimization of polysaccharide extraction yield using response surface methodology indicated that combination of the ultrasonic power of 58.06 percent (~337 W), extraction temperature of 65.15 °C and extraction time of 21.72 min resulted in maximum extraction yield (17.71%). Among three independent variables, ultrasonic power had the highest and temperature had the lowest impact on the rate of extraction. The results from antioxidant activity evaluation showed that even though extracted polysaccharide had lower absorbance capacity of the free radicals in comparison with control samples (ascorbic acid and BHT), but it revealed an acceptable antioxidant property.