The intricate mechanisms of iron (Fe) enhancing the simultaneous nitrogen (N) and phosphorus (P) removal in the denitrification biofilter (DNBF) remain enigmatic and warrant further investigation. Fe2+ and Fe3+ were added to compare the enhancing performances and the dosage concentration was optimized in this study, and the effects of Fe3+ on microbial characteristics were elucidated furtherly. Results demonstrated that the N removal performance with Fe3+ enhancing was better than Fe2+, and the optimal FeCl3 concentration was 0.56mg/L. The effluent concentrations of total phosphorus (TP), total nitrogen (TN), and chemical oxygen demand (COD) lowed to 0.27, 4.2, and 13.4mg/L, respectively. Fe3+ promoted microbial richness and diversity, with Proteobacteria, Bacteroidetes and Firmicutes emerging as the advantageous phyla for N and P removal in the DNBF. Microorganisms facilitated Fe3+ reduction to enhance denitrification process, and P was predominantly removed through the precipitation formed by the interaction of Fe and P. Furthermore, Fe3+ was noted to promote functional metabolism, thereby aiding in N removal. This study can reveal the effect of Fe3+ on N removal in the DNBF, and provide a theoretical foundation for advanced N and P removal.