Background Nowadays, microbial electrochemistry is widely used to produce valuable products (CH4, CH3CH2OH, CH3COOH, etc.) from carbon dioxide. Meanwhile, given that low‐value nitrogen (N2) is abundantly available as a feedstock for chemicals, thus ammonia (NH3) synthesis using bioelectrochemistry is still a promising technology. However, considering N2 stability and ammonium (NH4+) adsorption with a proton exchange membrane, developing suitable microbial systems for NH3 synthesis still faces challenges. Based on this, we developed a single‐chamber biocathode bioelectrochemical system for efficient NH3 production. Results For the reaction system that we constructed, NH4+ production reached 6.31 mg L−1 within 10 days, and NH4+ production was positively correlated with current density. A C2H2 reduction assay further verified the activity of nitrogenase enzyme. Microbial community analysis revealed that Clostridia plays a crucial role in the N2 fixation process, and the synergistic interaction between different microorganisms favors electron transfer and an increase in NH3 production. Conclusions The results of a series of experiments have proved that this green and economical method can be used to efficiently generate NH4+. This reaction system not only utilizes the advantages of obtaining electrons for reduction reaction, but also improves electron transfer and avoids the shortcomings of NH4+ adsorption. Additionally, this in‐depth study of N2 fixation mechanism provides a theoretical reference for other applications of bioelectrochemical systems. © 2022 Society of Chemical Industry (SCI).