Anaerobic co-digestion (ACoD) of poultry litter (PL) and wheat straw (WS) in an anaerobic sequencing batch reactor (ASBR) for continuous bio-energy generation was started up for the first time by gradually increasing the organic loading rate (OLR). A steady-state was reached with a daily biogas production of (13.06 ± 0.21) L and methane content of (54.38 ± 0.53) %. The subsequent regular operation achieved a daily methane yield of (100.41–188.65) mL CH4/g VS added and a total chemical oxygen demand (tCOD) removal rate of (70.3–85.9) % in the effluent under different operating parameters. The overall microbial community became more uniform, and the dominant aceticlastic methanogen of Methanosaeta was enriched after the start-up. While the microbial community was largely stable in the overall structure since the regular operation. Therefore, the start-up of the ACoD of PL and WS was successful with stable and continuous methane production.