Abstract: To solve the problem of high cost in traditional grass charcoal substrate breeding of Cyclobalanopsis gracilis seedlings, the feasibility of using Morchella esculenta mushroom residue as the main seedling substrate was explored.Taking decomposed M. esculenta mushroom residue as the main raw material, different proportions of peat, vermiculite, and perlite were added to make a composite substrates (Y1~Y9) with traditional peat soil culture medium as a control (CK). One-year-old seedlings of C. gracilis were used as experimental materials. Principal component analysis and membership function method were used to study the effects of various composite substrates on the growth of C. gracilis based on six indicators including seedling diameter increment, plant height increment,top dry weight, root dry weight, top/root ratio and sound seedling index. Comprehensive evaluation was conducted to screen the best substrate.The results showed that in the bacterial residue mixed matrix without added peat,five nutrient content indicators including total nitrogen, organic matter, available nitrogen, available potassium, and available phosphorus, as well as three physicochemical property indicators including aeration porosity, air-water ratio, and conductivity, gradually increased as the proportion of bacterial residue increased. There were varying degrees of correlation between the 12 matrix characteristic indicators and between characteristic indicators and growth indicators. Through comprehensive analysis of six growth indicators, it showed that except for Y2, Y3, and Y5, which had no significant differences or were significantly higher than the control in terms of various indicator values, all the other treatments had some indicator values significantly lower than the control.According to the comprehensive evaluation, Y2 (M. esculenta s mushroom residue: vermiculite: perlite = 4∶4∶2) had the highest comprehensive indicator value, followed by Y3 (M. esculenta s mushroom residue: vermiculite: perlite = 5∶3∶2). From the perspective of economic and ecological benefits, M. esculenta mushroom residue: vermiculite: perlite = 5∶3∶2 could more effectively save costs and reduce peat amounts so that could be recommended as the substrate for cultivatingC. gracilis seedlings.From the perspective of economic and ecological benefits, Y3 could more effectively save costs and reduce the amount of peat used, and could be recommended as a substrate for the cultivation of C. gracilis seedlings in production.