[Background] “Replacing wood by grass” is a concept to use other substrates than wood for edible fungi cultivation, to effectively alleviate the contradiction between edible fungi and forests. [Objective] To optimize a “replacing wood by grass” substrate formula by using different agricultural residues to replace sawdust. [Methods] In accordance to simplex lattice design method, 7 different agro-residues, namely wheat straw, corn straw, soybean straw, peanut straw, rape straw, rice straw and corncob were used for Pleurotus djamor cultivation. Enzyme activity of cellulase, polyphenol oxidase and laccase in the growth process of the mycelium was measured. The formulation suitable for the growth of Pleurotus djamor was analyzed and optimized. [Results] Soybean straw could significantly increase the mycelium growth of Pleurotus djamor, followed by wheat straw. Polyphenol oxidase activity of rape straw was highest. The optimized high mycelium growth formula includes (%, W/W), wheat straw 55.3, soybean straw 19.7, wheat bran 15.0, corn flour 5.0, bean pulp 3.0, lime 1.0 and gypsum 1.0. [Conclusion] A suitable substrate composed of various agricultural wastes instead of sawdust for mycelial growth of Pleurotus djamor was optimized for “replacing wood by grass” strategy.