Abstract: Pepper seedling substrate, mainly consisted of edible fungus scrap and cattle manure, was selected. In this study, with 13 kinds of seeding substrate formulas, in various proportions, mixed by edible fungus scrap, cattle manure and perlite, and with Lverwang seeding substrate (made in Shouguang, Shandong Province) mainly consisting of peat as control, the pepper variety,‘Hangjiao 5’was selected and complete random design was applied to carry out plug seedling. Consequently, it showed us that the seedling growth indexes treated by T₆ [cattle manure: mushroom stick: perlite=4:2:4 (V/V)], T₁₀ [cattle manure: mushroom stick: perlite= 3:5:2 (V/V)], T₁₁ [cattle manure: mushroom stick: perlite=3:4:3 (V/V)], were significantly better than the control. Besides, among them, plant height, stem diameter, fresh weight of the whole pant, aerial part and underground part, as well as dry weight of underground part of seedlings treated by T₁₀ had the best appearance, and those treated by T₆, T₁₁ both come second. In terms of cholorophyll content, seedling index, dry weight of aerial part and whole plant, T₆ had the best appearance and both of T₁₀, T₁₁ came second, however, the control had the lowest powder lump rate. In conclusion, comprehensively, T₆ and T₁₀ displayed a relatively better effect, thus, which could be considered as the optimal substrate formula for pepper seedling in Zhangye.