Two-chamber microbial fuel cell (MFC) with biogas slurry (BS) of corn stover as the anode substrate and Chlorella as the cathode substrate was investigated to solve the problem of the accumulation of wastewater generated from biogas plants and to achieve low-cost separation of CO2 from biogas. A simple two-compartment MFC was constructed using biocatalysis and inexpensive materials without expensive catalysts. The performance of MFC (X1-W, Y1-W, Z1-W) with different biogas solution concentrations as anode substrate and MFC (X2-C, Y2-C, Z2-C) with Chlorella as biocathode were compared, respectively. The MFCs (Z1-W,) can start quickly and maintain a stable power production (286.82 mV ± 184.59 mV). The growth rate of Chlorella at the MFCs (X2-C, Y2-C, Z2-C) biocathode was highly coincident with the output voltage. The MFC (Z2-C) has a maximum power density of 489.7 mW/m2 when the external resistance is varied to 200 Ω. The removal rates of chemical oxygen demand (COD) and ammonia nitrogen (NH3-N) are 93.42% and 92.59%. The maximum cell growth (Xmax) of Chlorella was 125.61 mg d-1, biomass productivity (Pmax) was 95.60 g L-1 d-1 and the maximum CO2 biofixation rate (RCO2) was 175.26 mg L-1 d-1. The microbial community analysis showed that the microorganisms in the anode solution were mainly from the biogas slurry and belonged to the hydrolytic bacteria. At the same time, the electroactive microbial community was mainly from anaerobic sludge. Therefore, MFCs can effectively degrade the organic matter in the biogas solution and generate electricity, and use Chlorella to absorb CO2 from the biogas, providing a new method for the development of biogas industry.