Abstract: Through the study on growth rate,culture characteristics and growth rules of Pisolithus tinctorius on different medium,the optimal medium was selected as the basic medium for its carbon and nitrigen sources study. The results show that Pt has a wide range of nutritional sources for metabolization,not only can it use monosaccharides,disaccharides,and inorganic nitrogen, but also it can use polysaccharides, polyol and organic nitrogen. Corn meal and mannitol are its optimal carbon sources, peptone and NH4Cl the optimal nitrogen sources for its mycelial growth. No obvious difference was noted when amine and nitrate forms of inorganic nitrogen were utilized by the fungus. And the optimum formulae of nutrition for its mycelial growth are obtained through orthogonal experiment L16(45).Through using the formulae,the biomass of the fungal mycelium can astonishingly proliferate 860 times,which surpass that of the former report. Therefore,the research provided the theory base to improve the formula and the technology for producing the ectomycorrhizal inocula of the fungus.