Spent mushroom substrate (SMS) is a potential biomass material generated during mushroom cultivation. In this study, the methane yield and microbial community resulting from co-digestion of SMS and dairy manure (DM) at different mixing ratios (0: 4, 1: 1, 3: 1, and 1: 3), were evaluated. Co-digestion analysis showed that the methane yield from the mixtures was 6%-61% higher than the yield from SMS or DM alone, indicating a synergistic effect of co-digestion of SMS with DM. For the SMS of F. velutipes (SFv) and P. erygii var. tuoliensis (SPt), co-digestion of DM/SMS at a ratio of 1: 1 was optimal, but for the SMS of P. eryngi (SPe), co-digestion of DM/SMS at a ratio of 3: 1 was ideal. The pH at all co-digestion ratios was in the range of 6.8-8.0, indicating that adding DM could increase the systemic buffering capacity. Methanosaetaceae was shown to be the predominant methanogens present during the co-digestion of DM/SMS.