Dairy manure (DM), chicken manure (CM) and wheat straw were used to investigate the possibility of optimizing the methane (CH4) potentials in anaerobic co-digestion of multiple substrates. Response surface methodology (RSM) was used to evaluate the individual and interactive effects of four variables [carbon/nitrogen (C/N) ratio, the feeding composition (DM/CM), initial substrate loading and inoculum-to-substrate ratio (ISR)] in the digestion process. All four variables had significant effects on CH(4) potentials. Interactive effects of C/N and DM/CM ratios, C/N ratio and ISR, initial substrate loading and ISR were significant The optimum conditions were a C/N ratio of 26.31, a DM/CM ratio of 42.96:57.04, an initial loading of 15.90 g volatile solids (VS)/l and an ISR ratio of 2.34, with the maximum CH4 potential being 394 ml/g VS. The RSM model was appropriate for optimizing CH4 production in the process of anaerobic co-digestion of multiple substrates.