[Background] Polysaccharide is one of the main substances in Hericium erinaceus and is commonly used in the treatment of neurasthenia, tumors, and digestive diseases like gastritis and ulcers. At present, studies on polysaccharides from H. erinaceus mainly focus on the structural analysis, but few on the effects of different substrates on the physicochemical properties of polysaccharides from the fruit bodies. [Objective] The physicochemical properties and in vitro immune activity of polysaccharides from fruit bodies of H. erinaceus cultivated on different substrates were studied to select the better cultivation formula of H. erinaceus with high yield and good quality. [Methods] Different fruiting bodies of H. erinaceus were obtained by industrial cultivation with seven different formulations of substrates. The yield of fruit bodies and the content of crude polysaccharides were analyzed and determined. The physicochemical characteristics such as molecular weight distribution and monosaccharide composition of the polysaccharides of H. erinaceus fruiting bodies obtained from seven different substrate cultures were analyzed, and the differences in NO release from RAW264.7 macrophage cell strains were compared. [Results] All indicators showed that the formula 2 (sawdust 30%, corncob 40%, cottonseed shell 15%, corn flour 2%, wheat bran 6%, rice bran 5%, gypsum 1%, lime 1%) and formula 7 (corn cob 39%, cottonseed shells 10%, wheat bran 10%, rice bran 30%, soybean bran 5%, beet residue 4%, calcium carbonate 2%) can be considered as excellent, efficient and high quality cultivation compared with other formulations of culture substrates. [Conclusion] The selected culture formulas are suitable for the cultivation fruiting body of the deep processed raw materials for H. erinaceus.