Abstract: Objective:To investigate the effect of extraction methods on the antioxidant activity of crude polysaccharide from Opuntia Milpa Alta Haw in vitro. Methods:Polysaccharides in fresh Opuntia Milpa Alta Haw were extracted by the methods of hot water extraction,enzymatic extraction and microwave extraction respectively,then use the phenol-sulfuric acid method to determine the purity of polysaccharides. The antioxidant activities of the extracted polysaccharides were analyzed by the anti-lipid peroxidation on spontaneous hepatic tissue,the clearance of hydroxyl radical,superoxide anion and DPPH·,total reduction,total antioxidant capacity. Results:The polysaccharides prepared by enzymatic extraction had the highest extraction yield 10.14%,while the microwave extraction had the highest purity 47.62%,enzymatic extraction and microwave extraction had a higher clearance rate than hot water extraction in the whole antioxidant indicators. Conclusion:The antioxidant activity of polysaccharide in vitro was associated with extraction methods,and the polysaccharides extracted by enzymatic extraction had a better antioxidant activity.