The purpose of this study was to optimize the extraction method for polysaccharide from the fruiting bodies of Pleurotus ostreatus (Jacq.) P. Kumm and to assess the antioxidant and cytotoxic potentials of polysaccharide. In this investigation, polysaccharides from Pleurotus ostreatus (Jacq.) P. Kumm were extricated by utilizing the hot water. One-single factor and response surface methodology was established to optimize the extraction conditions for polysaccharide from Pleurotus ostreatus (Jacq.) P. Kumm. Examination of antioxidant activity of Pleurotus ostreatus polysaccharide (POP) was directed by utilizing 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azino-bis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) techniques. Cytotoxicity of POP was evaluated using an MTT assay. The experimental data were fitted to a quadratic equation utilizing multiple regression investigations, and the ideal conditions were as per the following: water/crude material proportion, 26.04 mL/g; an extraction time of 62.08 minutes; and an extraction temperature 70.5°C. Under such conditions, the polysaccharide yield was 5.32 ± 0.12% with the anticipated yield. POP showed good scavenging activity against DPPH radical (p<0.001, EC50 = 1036.38 μg/mL, R2 = 0.8313) and ABTS radicals (p<0.001, EC50 = 824.37 μg/mL, R2 = 0.8223), with a dose (p<0.001)-and-time (p<0.001) dependent cytotoxic potential on Ehrlich ascites carcinoma cell line in vitro. This demonstrated that polysaccharides (POP) had certain cancer prevention agent exercises. In this manner, these examinations give reference to additionally research and reasonable improvement of Pleurotus ostreatus (Jacq.) P. Kumm polysaccharide and POP may prove a useful therapeutic agent, due to its robust antioxidant and cytotoxic activity.