A rectangular double chamber with trivalent arsenic as the electron donor of the biological anode was constructed by microbial fuel cells (MFC), and the feasibility of the MFC simultaneous degradation of trivalent arsenic and nitrate was studied. Experimental results show that the co-matrix-coupled MFC reactor oxidizes trivalent arsenic in an anode chamber and degrades nitrate in the cathode chamber. The removal rate of trivalent arsenic is about 63.35%, and the degradation rate of nitrate is about 55.95% during the complete and stable operation period. MFC can continuously output electric energy, and the maximum output voltage is 388 mV. We compared and analyzed the main functional microflora of biofilm microorganisms in an anode chamber. In the long-term arsenic-polluted environment, the activity of Acinetobacter, Pseudomonas bacteria with arsenic resistance, was improved. It is inferred that a fraction of trivalent arsenic was oxidized to pentavalent arsenic by electrode-attached microorganisms. While remaining trivalent, arsenic was taken up by the suspended bacterial biomass and converted into stable arsenide. The results of this study have theoretical reference value for the expansion of the MFC application scope.