The efficient removal of nitrate nitrogen (NO3–N) and reduction of greenhouse gas (GHG) emissions in bioretention system (BRS) poses challenges. This study aimed to investigate the performance of a pyrite/biochar-based BRS coupled with microbial fuel cell (MFC) (PBM-BRS) in terms of pollution removal, bioelectricity generation, and GHG emissions under various operating conditions. Compared with the conventional bioretention system (C-BRS), the PBM-BRS demonstrated an enhanced capacity for pollutant removal, with an increase of 5%-30%. It also exhibited low GHG emissions, as evidenced by a CO2 equivalent (CO2eq) release flux of 971.20 +/- 277.54 mg CO2/(m(2)d). As the influent C/N ratio increases, all systems experienced an increase in NO3–N and TN removal, output voltage, and power density. Meanwhile, the accumulation of nitrite nitrogen (NO2–N) gradually diminished, and the emission fluxes of CO2eq decreased significantly (P < 0.05). Microbial analysis revealed that the PBM-BRS significantly affected the community structure, promoting the proliferation of electroactive bacteria (e.g., Geobacteraceae), and augmenting the abundance of denitrification functional enzymes (narG, nirS, norB, and nosZ) through the incorporation of pyrite. The SEM and spectroscopy (XPS and FTIR) results indicated that the electrochemical action facilitated Fe2+/Fe3+ in-situ supported on biochar (Fe@BC), promoting electron transport. This process impeded the respiration of methanogenic bacteria and diminishing the formation of intermediate product N2O. In conclusion, the PBM-BRS demonstrated superior performance in the removal of NO3--N, reduction of GHG emissions, and bioelectricity generation through a synergistic action of adsorption, electrolysis, and microbial degradation. This confirms the high potential of PBM-BRS as a highly promising technology for water treatment.