Abstract: A new repeated freeze-thaw extraction was employed to extract crude polysaccharide from Ganoderma lucidum, named as GLPf, in order to improve its extraction yield and bioactivity. Single-factor tests and response surface experiment were used to obtain the optimal extraction conditions. Three novel G. lucidum polysaccharide fractions (GLPf30, GLPf60, and GLPf80) were isolated and purified from GLPf by stepwise alcohol precipitation. Results showed that the optimal parameters were as follows: Swelling ratio 1:14 (g/mL), free-ze-thaw time 140 min, and free-ze-thaw times was 3. Under these conditions, its extraction rate was 2.71%, which was significantly higher than that of polysaccharide extrcted by traditional water extraction (GLP, 2.36%). The preliminary structural features of all polysaccharide fractions were determined by highperformance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD) and Fourier transform infrared spectrophotometer (FT-IR) analyses, which showed that all sugar fractions were acidic polysaccharides, and they all were composed of six monosaccharides (fucose, glucosamine, galactose, glucose, xylose and mannose) with pyranose as the backbone. However, there were significant differences in both monosaccharide composition and physicochemical property, and GLPf30 had the highest percentage of GlcN (up to 37.66%). The results of antioxidant activity assays showed that antioxidant power of GLPf30 was significantly higher than that of the other two polysaccharides (GLPf60 and GLPf80) and their precursors GLPf. These findings would provide scientific basis for the rational development and high value utilization of G. lucidum polysaccharides.