The quadratic orthogonal rotation regression combination design was employed in this study to determine effects of poultry manure amount, biochar dosage and C/N ratio on biocharmediated mesophilic anaerobic digestion of poultry manure. These three factors were then subjected to response surface methodology to obtain optimum parameters for methane production. The results showed that specific cumulative methane production (SCMP) could be obviously increased by adding appropriate amount of biochar during anaerobic digestion of poultry manure. SCMP was increased as biochar dosage increased from 2.5% to 5.0%, but was significantly decreased with the further increase of biochar dosage up to 7.5% (P<0.05). Lower poultry manure amount could result in higher SCMP. The effect of C/N ratio on SCMP showed the same trend with biochar dosage, namely an initial increase up to certain level and followed by decreasing change. The highest SCMP (288mL/g) was observed with biochar dosage of 5.0%, poultry manure amount of 17.83g and C/N ratio of 25. The developed mathematical regression model here showed a good fit with the experimental data. Moreover, the model predicted that the maximum SCMP could be obtained with biochar dosage of 4.97%, poultry manure amount of 14.35g and C/N ratio of 22.02.