Biogas yield from anaerobic digestion of cotton stalk (CS) at feed to inoculum (FP) ratios of 2-4 reached 175-180 mL/(g of VSadded) against 113 mL/(g of VSadded) observed at the F/I ratio of 6. CS was proven to be a promising co-substrate in the digestion of swine manure (SM), and a CS/SM ratio of 50:50 with a C/N ratio of 25 was found to be the best in terms of the biogas production rate and yield with increases up to 1.8- and 1.9-fold, respectively, as compared to the control. The highest biogas yield (449 mL/(g of VSadded)) and production rate (0.65 L/L.day)) with comparable technical digestion time were obtained in co-digestion of SM with CS pretreated by NaOH, which was 241-255% of those achieved in the control. This study indicates that co-digestion of SM with alkali-pretreated CS is a potential option for alleviating the insufficient substrate resource problems and improving the energy output.