The aim of this study was to evaluate the efficiency and optimization of co-digestion using sewage sludge (SS), maize straw (MS) and cow manure (CM) as feeds, and the effects of the mixing ratio and C/N ratio of the substrates were analyzed in detail. Among the three substrates tested, CM/MS exhibited better digestion than CM/SS and SS/MS in terms of all measures, including total daily biogas and net methane volume production, due to the hydrophilic characteristics and high level of biodegradability of CM, as well as its higher C/N ratio. The average biogas production was 613.8 mL/g VS for the co-digestion of CM/MS at a feed concentration of 15 g VS/L and using a 1:1 mixing ratio (C/N ratio of 28.3). The co-digestion of SS/CM/MS performed better than the individual digestion of the components because of the balanced C/N ratios and supply of carbon. The optimum conditions for maximizing methane potential were an SS:CM:MS ratio of 30:35:35 and a bulk VS concentration of 15.0 g VS/L, which led to a maximum methane production of 8047.31 mL (C/N ratio of 12.7). The high-throughput sequencing analysis showed clear differences in microbial communities during the entire co-digestion process.