Aiming to optimize the methane production efficiency of semi-continuous anaerobic digestion of corn stover, three types of mixed gas nanobubble water ( NBW) were prepared: ( H2 + CO2 ) - NBW (volume ratio = 4∶1), (CO2 + CH4) - NBW (volume ratio = 2∶3), and (CO2 + H2 + CH4) - NBW (volume ratio = 4 ∶1 ∶5). The effects of different mixed gas NBW on methane production, substrate degradation, thermophysical properties of digestate, and microbial community structure were investigated. The results showed that compared with the control group, the average daily methane yield was increased by 15.76% , 4.51% , and 6.87% in the (H2 + CO2) - NBW, (CO2 + CH4 ) - NBW, and ( CO2 + H2 + CH4 ) - NBW groups, and the maximum methane volume fraction was increased by 8.01% , 5.20% , and 8.37% , respectively. In terms of substrate degradation, mixed gas NBW promoted higher concentrations of volatile fatty acids ( VFAs), reducing sugars, and soluble chemical oxygen demand (SCOD) in the early stage of semi-continuous operation. Compared with the control group, the total solid (TS) removal rates were enhanced by 40.10% , 10.82% , and 16.36% in the 3 NBW groups, respectively. Meanwhile, mixed gas NBW effectively improved the heat transfer and flow characteristics of the digestate. The average thermal conductivity of the digestate was increased by 2.93% , 2.87% , and 0.98% in the (H2 + CO2) - NBW, (CO2 + CH4) - NBW, and (CO2 + H2 + CH4) - NBW groups, respectively, while the average apparent viscosity was decreased by 10.51% , 14.89% , and 14.79% , respectively. Microbial community analysis revealed that the introduction of mixed gas NBW increased the relative abundance of hydrogenotrophic methanogens ( e. g. , Methanolinea, Methanospirillum, Methanomassiliicoccus) and hydrolytic and acidogenic bacteria ( e. g. , ADurb. Bin120, Aminicenans, Clostridium). These findings can be expected to provide theoretical basis and data support for the industrial application of nanobubble technology in anaerobic digestion.