Abstract: In this study, using Phellinus igniarius as raw materials, the extraction conditions of Phellinus igniarius polysaccharides were optimized, the structure and immune regulatory activity were analyzed and evaluated. Using extraction temperature, extraction time, solid-liquid ratio, and extraction frequency as factors, single factor experiments and orthogonal experiments were used to optimize the extraction conditions of polysaccharides from Phellinus igniarius. After purification by enzymolysis and dialysis methods, the structural features of this polysacharide were characterised using HPSEC (Mw), FTIR (functional group), HPAEC (monosaccharide composition) and methylation analysis (linkage patterns), and their immunomodulatory properties were investigated using RAW-Blue™ cell line. Results showed that, the optimal extraction conditions of polysaccharides from the fruiting body of Phellinus were as follows: The solid-liquid ratio was 1:40 (g/mL), the extraction temperature was 100 °C, the extraction time was 2.0 h, and the extraction times were 3. Under these conditions, the extraction yield of polysaccharide was 6.71%, the total sugar content was 81.69%±0.19%, and the Mw was 10.77 kDa. The polysaccharide obtained belong to glucan family constructing mainly by (1→4)-Glc, small percentage of (1→6)-Glc and (1→3)-Glc as side chains. This polysaccharide could enhance the embryonic alkaline phosphatase secretion activity and the phagocytosis ability of RAW-Blue™ macrophages, which would provide a theoretical basis for its application in both food and phamacetical area.