The syngas produced from biomass gasification is a great potential energy resource, which can well be utilized to produce biomass-based substitute natural gas (BioSNG) via syngas biomethanation. CO biomethanation is one of the key issues in the biomethanation process and was studied experimentally in this work with respect to the effect of anaerobic granular sludge semi-disaggregation. The results show 1.07 times higher averaged CH4 production rate with the semi-disaggregated granular sludge than the whole granular sludge at 35 °C, and 1.69 times higher at 55 °C. The main mechanisms behind the enhanced CH4 production rate, especially under the thermophilic condition, are the improvement of microbial interspecific syntrophic association caused by the higher electron and substrate transfer rate, and more active cell growth and metabolism as reflected in higher abundance of functional genes and enzymes and less useless extracellular polymeric substances. The CO biomethanation enhancement occurs in the conversion of the substrate to the intermediate products. The semi-disaggregation of anaerobic granular sludge or similar way to strengthen interspecific association is an effective approach to improve the ability and tolerance of microbial cultures under the CO atmosphere. This technique can well be applied for the energy conversion from the CO-rich gas substrates into BioSNG via CO biomethanation under the thermophilic condition, or for the production of intermediates as fuels/chemicals under the mesophilic condition.