This article focused on the research of anaerobic digestion conditions using low-temperature pyrolysis oil, including the fresh inoculum acclimatization, the operation conditions of pyrolysis oil (PO) digestion and the influence of biomass pyrolysis parameters. The coupling process was studied by controlling different pyrolysis parameters and anaerobic digestion parameters. It can be concluded that the fresh sludge inoculum acclimatization can significantly improve the tolerance to the inhibitors contained in the PO, thus, the methane production from the PO digestion to a great degree. The mesophilic condition was favorable to the biooil biomethanation for the low POs under 4% as used in the experiment, whereas the thermophilic condition was favorable for the high PO concentration of 10%. Besides, more methane production can be given by 0.85mm biomass particle size, 300℃ pyrolysis temperature in the downstream step of PO digestion.