Abstract: To clarify how substrate composition influenced the nutritive profile and protein nutritional value in Stropharia rugosoannulata under selenium enrichment, the nine agricultural by-products were used to cultivate Stropharia rugosoannulata, rice straw, corn stalks, corn cobs, weeds, peanut stalks, Lentinus edodes residue, Pleurotus ostreatus residue, Auricularia auricula waste substrate and bamboo leaves. The results showed that selenium-enriched cultivation significantly increased both yield and selenium concentration of Stropharia rugosoannulata (P<0.05). Specifically, the selenium content of Stropharia rugosoannulata cultivated with raw corn cobs was the highest, reaching 154.41 mg/kg, which represented a 203.53-fold increase compared with the non-enriched control grown on the same substrate. Significant variations were observed in the nutritional quality of selenium-enriched Stropharia rugosoannulata cultivated with different substrates. Selenium-enriched Stropharia rugosoannulata produced on corn cobs and weeds contained significantly more crude fat (P<0.05), whereas those cultivated on corn stalks and bamboo leaves showed higher crude protein but lower crude fibre (P<0.05). Regarding protein quality, fruiting bodies from spent Pleurotus ostreatus residue exhibited an essential amino-acid pattern closest to the WHO/FAO reference and the highest SRCAA (amino-acid ratio coefficient score). The highest AAS (amino acid score) and CS (chemical score) were obtained with mushrooms grown on Auricularia auricula waste substrate. The comprehensive evaluation showed that the selenium-enriched product from Auricularia auricula waste substrate had the highest protein nutritional value, and selenium-enrichment cultivation improved its protein nutritional value. This study provides a theoretical basis for the application of selenium-enriched Stropharia rugosoannulata and its development as a new protein resource.