In order to separate and purify polysaccharides in Cordyceps sinensis and study its autoxidation. The extract effect factor of polysaccharides in Cordyceps sinensis such as extraction time, temperature, solid/liquid ratio, were investigated based on the content of polysaccharides. The interactions of time-temperature, temperature-solid/liquid ratio and time- solid / liquid ratio, etc were investigated by response surface methodology. The polysaccharide was purified by Sepharose CL-6B gel filtration. Meanwhile, antioxidation of purified polysaccharides was studied. The Design Expert software was employed to inversely resolve the created quadratic regression model of the extraction yields of polysaccharides. The optimums conditions were as follows: the extraction time was 2 h, solid/liquid ratio was 1:34 and temperature was 100℃. The purified polysaccharides in Cordyceps sinensis showed stronger antioxidants. It could remove scavenging DPPH free radical and superoxide radical were investigated respectively.