Abstract: The hybrid system integrating a microbial fuel cell and constructed wetland (MFC-CW) utilizing pyrite substrates demonstrates the ability to enhance autotrophic denitrification. By leveraging reducible organic electrons, the system achieves a synergistic effect of autotrophic and heterotrophic denitrification in mixotrophism, particularly when treating wastewater with a low carbon to nitrogen ratio. In this study, we compared the denitrification rates in a pyrite-based dual-anode MFC-CW under different carbon to nitrogen ratios (0 and 2.5) and initial nitrate concentrations (7, 14, and 28 mg/L) at upper and lower anodes. Additionally, the study simulated the kinetics of the nitrate reduction reaction at different stages to elucidate the autotrophic-heterotrophic synergistic denitrification mechanism from a kinetic perspective. The results showed that: the Denitrification efficiency at the two anodes did not significantly differ under different carbon and nitrogen ratios, but variations were observed in nitrite accumulation and sulfate generation; Microbial community composition at the two anodes exhibited similarity, with the relative abundance of dominant genera significantly influenced by the C/N ratio and anode position. Nitrate reduction kinetics at both anodes represented a first-stage reaction, with the denitrification rate constant being smaller at C/N=2.5 compared to C/N=0; The denitrification kinetics of MFC-CW system aligned more with the Monod-CSTR model, with denitrification effect concentrated in the first 6h. In conclusion, the denitrification kinetics of the pyrite-based MFC-CW system, following the Monod-CSTR model, underscore its effectiveness, especially within the initial 6h. These results can provide theoretical guidance for the practical engineering application of pyrite-based MFC-CW.