Abstract: This study aimed at evaluating the antioxidant activity in vitro and vivo of polysaccharide prepared from Paulownia fortunei flower by water extract-alcohol precipitation. In vitro, results showed that Paulownia fortunei flower polysaccharide had a dose-dependent and good effect on scavenging superoxide anion, hydroxyl radical and inhibiting H₂O₂ hemolysis. When the concentration of polysaccharide controlled at 0.5 mg/mL, the scavenging rates of superoxide anion and hydroxyl radical were 54.36% and 74.62% respectively, with more than half effect of 50%. When at 2 mg/mL, the anti-H₂O₂ oxidation hemolysis rate was higher than that of the positive control Vc group (1 mg/mL). In vivo, after the experimental mice were administered to oral gavage with Paulownia flower polysaccharide at low, medium and high dosages (5, 10 and 20 mg/mL, 0.2 mL) for 28 consecutive days. The result showed that Paulownia fortunei flower polysaccharide had no effect on weight of mice; different doses of polysaccharide could enhance or siginificantly enhance the content of superoxide dismutase (SOD), glutathione (GSH), total antioxidant capacity (T-AOC) remarkably in serum, heart, liver, kidney, ileum (P<0.05) when compared with that of the blank group, and reduce the malondialdehyde (MDA) content dramatically with a certain concentration-response relationship. The results indicate that Paulownia fortunei flower polysaccharide had perfect antioxidant activity and could be used as a natural antioxidant in food and medicine industry.