Direct interspecies electron transfer (DIET) in microbial communities plays a significant role in improving efficiency of biomethane production from anaerobic digestion. In this study, the impacts of conductive graphene on mesophilic and thermophilic anaerobic digestion (MAD and TAD) were comparatively assessed using the model substrate ethanol. The maximum electron transfer flux for graphene-based DIET was calculated at mesophilic and thermophilic temperatures (35 °C and 55 °C). Biomethane potential results showed that the addition of graphene (1.0 g/L) significantly enhanced biomethane production rates by 25.0% in MAD and 26.4% in TAD. The increased biomethane production was accompanied with enhanced ethanol degradation. The theoretical calculation for maximum DIET flux showed that graphene-based DIET in MAD (76.4 mA) and TAD (75.1 mA) were at the same level, which suggests temperature might not be a significant factor affecting DIET. This slight difference was ascribed to the different Gibbs free energy changes of the overall DIET reaction (CH3CH2OH + 1/2CO2 ¿ 1/2CH4 + CH3COO- + 5H+) in MAD and TAD. Microbial analysis revealed that the dominant microbes in response to graphene addition were distinctly different between MAD and TAD. The results indicated that the bacteria of Levilinea dominated in MAD, while Coprothermobacter dominated in TAD. The abundance of archaeal Methanobacterium decreased, while Methanosaeta increased with increasing temperature.