Abstract: In order to improve the extraction rate of crude polysaccharide from Schizophyllum mycelium and explore the biological activity of purified intracellular polysaccharide, this study adopted the method of ultrasonic-assisted hot water extraction to extract intracellular crude polysaccharide from Schizophyllum mycelium and the extraction process was optimized by response surface method. On this basis, DEAE-52 ion-exchange column and Sephadex G-100 dextran gel column chromatography were used to separate and purify the crude polysaccharide, and then the structural characterization, antioxidant capacity and antibacterial activity of the purified polysaccharide were carried out. Results showed that the optimal extraction conditions of intracellular crude polysaccharides were as follows: Under the extraction temperature of 90 ℃, the water-material ratio 30:1, the ultrasonic time 30 min, the ultrasonic power 230 W. Under these conditions, the extraction rate of intracellular crude polysaccharide was 18.14%±0.33%. The purified polysaccharide fraction NSPG-1 was a β-pyranose sugar with the molecular weight of 1.05×10⁶ Da. NSPG-1 had good antioxidant capacity and the IC₅₀ values for DPPH radical, hydroxyl radical and superoxide anion radical were 6.97 1.07 and 11.41 mg/mL, respectively. NSPG-1 had good antibacterial activity and the IC₅₀ values against Escherichia coli, Bacillus subtilis and Staphylococcus aureus were 7.56, 12.54 and 10.42 mg/mL, respectively. The results would provide a basis for the industrial production and utilization of Schizophyllan.