To improve the nitrogen removal capability of constructed wetlands, the biochar, produced from bamboo, activated with HCl and coated with Fe (FeCl3·6H2O), and then was added as a substrate into the systems. Three horizontal subsurface flow constructed wetlands (HSCWs) was established to treat the tailwater from the wastewater treatment plant: C-HSCW(quartz sand+soil), B-HSCW(quartz sand+soil+unmodified biochar), and FeB-HSCW(quartz sand+soil+Fe-modified biochar). Under different combinations of hydraulic retention time and nitrogen loading, the FeB-HSCW revealed extremely effective nitrogen removal. The highest removal efficiencies of NO3−-N (95.30%), Total N (86.68%), NH4+-N (86.33%), NO2−- N (79.35%) were obtained in FeB-HSCW with the hydraulic retention time of 96 h. and low influent nitrogen loading. Nitrogen mass balance analysis showed that microbial processes played the most important role of nitrogen removal in HSCWs and the Fe-modified biochar significantly enhanced the microbial nitrogen removal. The contributions of microorganisms, substrate storage and plant uptake on the total amount of nitrogen removal in the FeB-HSCW was 92.69%, 2.97% and 4.34%, respectively. Moreover, FeB significantly increased the abundances of genes involved in nitrogen removal. Thus, Fe-modified biochar provides a feasible and effective amendment for constructed wetlands to improve the nitrogen removal, particularly nitrate-N, for low C/N wastewaters by enhancing the microbial nitrogen removal capacity.