Taking substitution rate as an index, the effects of concentration of sulfuric acid, reaction temperature and reaction time on sulfation of Pleurotus ostreatus polysaccharides were studied by concentrated sulfuric acid method. On the basis of single-factor results, the optimum preparation process of P. ostreatus polysaccharide sulfate was obtained by L₉(3⁴) orthogonal test. Secondly, the polysaccharide and sulfates of P. ostreatus polysaccharide were characterized by FT-IR spectrometer. Finally, the reductive power of V_C and the scavenging rates of DPPH radical, superoxide anion radical and hydroxyl radical were determined to explore the antioxidant activity of P. ostreatus polysaccharide sulfate. The optimum preparation process of polysaccharide sulfate of P. ostreatus was as follows: the mass of P. ostreatus polysaccharide was 1.00 g; the volume of sulfuric acid was 10 mL, reacting at 60 ℃ for 90 min, and the degree of substitution was 0.48. FT-IR spectrum results showed that sulfate group and P. ostreatus polysaccharide formed polysaccharide sulfate. The IC₅₀( scavenging rate) of polysaccharide sulfate from P. ostreatus were 0.816, 1.882 and 1.611 mg/mL for DPPH radical, superoxide anion radical and hydroxyl radical, respectively. The reductive power and scavenging ability to three free radicals of P. ostreatus polysaccharide sulfate were higher than those of polysaccharides, which suggested that P. ostreatus polysaccharide sulfate had a better antioxidant activity than that of polysaccharides.