Auricularia polytricha was cultivated on a sawdust basal substrate supplemented with different proportions (30%, 45%, and 60%, respectively) of stalks of three grass plants, i.e., Panicum repens (PRS), Pennisetum purpureum (PPS), and Zea mays (ZMS), to determine the most effective substrate. The mycelial growth rate, total colonization time, days to primordial formation, biological efficiency and chemical composition of fruiting bodies were evaluated. The results indicated that 30PPS was the best substrate for mycelial growth of A. polytricha, with a corresponding total colonization period of 32.0 days. With the exception of 30PPS, the total biological efficiency of all of the substrates containing P. repens stalk, P. purpureum stalk and Z. mays stalk was higher (P < 0.05) than that of the control. The most suitable substrate with a high biological efficiency was 60PRS (148.12%), followed by 30ZMS (145.05%), 45ZMS (144.15%) and 30PRS (136.68%). The nutrient values of fruiting bodies were affected by different substrates. The ash contents of A. polytricha cultivated on a substrate containing Z. mays stalk were higher than that of the control; meanwhile, the protein contents of mushroom cultivated on a substrate containing P. repens stalk (except substrate 45PRS) were higher than that of the control. The biological efficiency of the substrates was tested, and according to the results, it is feasible to use the stalks of P. repens and Z. mays on partially replaced sawdust to cultivate A. polytricha.