Abstract: In order to improve the antioxidant activity of Zizania latifolia polysaccharides, sulfate it and explore the optimum modification conditions. Polysaccharides was extracted by water extraction and alcohol precipitation method from Zizania latifolia, then it was separated and purified to obtain a new type of water-soluble polysaccharide. Using formamide as solvent, polysaccharides were sulfated by sulfur trioxide-pyridine method. The degree of substitution (DS) was set as the examination index, the optimal sulfonation conditions were selected by response surface method. Through the free radical scavenging rate trial in vitro, compared with natural polysaccharides, to explore the changes of antioxidant activity of sulfated polysaccharides in vitro. The results showed that the optimum reaction conditions obtained as polysaccharide 100 mg, time 4 h, temperature 70 ℃, sulfur trioxide-pyridine complex 2.6 g. The optimum DS was 1.79. Thermogravimetric analysis showed that the fastest weight loss rate of Zizania latifolia polysaccharides was 16.48% at 46.38 ℃, and the sulfated polysaccharides was 2.92% at 65.04 ℃. In the antioxidant test in vitro, the IC₅₀ of free radical scavenging ability of Zizania latifolia polysaccharides to DPPH·, ·OH and ABTS⁺· were 3.49, 3.28 and 12.70 mg/mL respectively. The sulfated polysaccharides of Zizania latifolia polysaccharides were 0.39, 1.00 and 1.82 mg/mL respectively, and the positive control (V_C) was 0.069, 0.17 and 0.077 mg/mL respectively. In summary, sulfation modification can enhance the thermal stability of Zizania latifolia polysaccharides and enhance its free radical scavenging ability to some extent.