Abstract: To rationally utilize the resources of Coix straw, this study designed different ratios of Coix straw formulations to cultivate Grifola frondosa, comparatively analyzed the differences among agronomic indices, protein and crude polysaccharide content of different formulations, and analyzed the composition and content of amino acids of G. frondosa on the based on the current international amino acid pattern profile using the amino acid score (AAS), amino acid ratio (RAA), ratio coefficient of amino acid (RC), chemistry score (CS), essential amino acid index (EAAI), biological valence (BV), and nutritional index (NI) for the comprehensive evaluation of protein nutrition of G. frondosa. The results showed that the relative biological conversion of formulation P3 (30% Coix straw, 10% cottonseed husk, 30% fine sawdust, 18% bran, 5% cornmeal, 6% clay, and 1% gypsum) was increased to 31.25%±1.02%. The protein content of 33.36 g/100 g was higher than that of the control group of 6.76 g/100 g. Evaluation of the nutritional value of AAS, RAA, RC, and CS showed that the first limiting amino acid of G. frondosa was methionine (Met). The EAAI, BV, and NI values of the P3 formulation were 105.06, 102.82, and 35.05, respectively; all three index scores were better than those of the CK formulation, and the values were better than those of the model protein. In conclusion, the amino acid distribution of G. frondosa cultivated by formula P3 was balanced, with high nutritional value and prospects for healthy food development. The present study was conducted to provide a theoretical basis for the efficient cultivation of high-quality G. frondosa with Coix straw and the necessary methods for efficient resource utilization of Coix straw.