By optimizing fillers properties, packing modes, DO concentration and HRT, the NH4+-N, NO3--N and PO43--P coinstantaneous removal was achieved by BAF, and the effluent concentration of TN and PO43--P were below 1.2 and 0.2 mg/L with DO and HRT maintaining at 1.2–1.5 mg/L and 8 h, respectively. Kinetic analysis indicated that sulfur and calcined pyrite mixed packing in BAF has more excellent providing electron capability and nutrients removal efficiency. Furthermore, the Thiobacillus, Ferritrophicum, Simplicispira, Nitrosomonas and Nitrospira as main nitrification and autotrophic denitrification genera were simultaneously enriched. Meanwhile, more EPS and signal molecules were secreted under Fe2+ stimulation. In batch experiments, EPS could accelerate electron transport efficiency via increasing electron transport system activity (ETSA) and decreasing electrochemical impedance spectroscopy (EIS), and EPS also contributed to biofilm formation and stability via improving Zeta potential and relative hydrophobicity. Additionally, the OD600 results showed that the signal molecules could regulate the bacteria density and indirectly affect the nitrogen removal. Generally, the variations in the macro-environment and feedback effect of EPS and signal molecules were important factors to achieve efficient nutrients removal.