Abstract: Objective: To investigate the purification of polysaccharides from Opuntia Milpa Alta and the antioxidant activity in vitro. Methods: Crude polysaccharides from Opuntia Milpa Alta ( MAP) was extracted by water extraction and alcohol sedimentation.Two polysaccharides named MAP1 and MAP2 were achieved after MAP was purified through DEAE-cellulose-52, and another polysaccharide named MAP3 was achieved through DEAE-Sepharose-CL-6B. The relative molecular mass of polysaccharides was analyzed by gel permeation chromatography ( GPC) and the antioxidant activity was investigated in vitro.Results: The relative molecular mass ( Mn) of MAP1 was 476.9 ku, MAP2 contained three relative molecular mass ( Mn) 5449, 9724, 529 ku, MAP3 contained 13270, 15.87, 474 ku. The results of antioxidant activity in vitro showed that, MAP had the highest total antioxidant capacity, the activity of MAP2 and MAP3 were higher than MAP in scavenging hydroxyl radical, superoxide anion and DPPH radical, and MAP2 had a higher clearance rate than MAP3 in the whole antioxidant indicators.Conclusion: The results indicated that MAP2 had a better antioxidant activity, while antioxidant activity of polysaccharide in vitro was associated with relative molecular mass.