The invention relates to sulphating grifola frondosa insoluble polysaccharide. A preparation method comprises the steps of taking grifola frondosa polysaccharide as a raw material, using N,N-dimethylformamide for swelling, adding sulfur trioxide trimethylamine, and obtaining sulphating grifola frondosa insoluble polysaccharide through water bath reaction, cooling, centrifugation, washing and dialysis. When the polysaccharide is at a certain concentration and time range, an S-GFP can remarkably improve proliferation and phagocytic activity of RAW264.7 cells and have dosage dependence, the optimal action time is 36 h, the optimal action dosage is 40 [mu]g/mL, and secretion and activity of cell acid phosphatase, lysozyme and superoxide dismutase are improved remarkably. The expression of RAW264.7 cell iNOS genes is induced and up-regulated at the mRNA transcriptional level, the generation amount of cell NO is increased, the secretion of cytokines such as IL-2, IL-12, IFN-gamma and IL-6 is promoted, and the sulphating grifola frondosa insoluble polysaccharide is high in immune enhancement activities and can be applied to preparation of medicines or health care products for enhancing immunity.