Abstract: To investigate the denitrification efficiency, nitrogen conversion pathways and corresponding microbial flora functions of bioelectrodes at different potentials, a sleeve-type bio-electrochemical system (BES) device with multiple working electrodes was constructed in this study. Three different constant potentials including -0.3 V, -0.5 V and -0.7 V were applied to the working electrodes simultaneously. Results show that the denitrification rates and current densities are 0.88 mg/(L·h) and 36.91 mA/m² at -0.3V, 1.02 mg/(L·h) and 241.92 mA/m² at -0.5 V, 1.11 mg/(L·h) and 476.41 mA/m² at -0.7 V, respectively. The maximum denitrification rate and current density are both obtained at -0.7 V, indicating that more negative potential is beneficial for microorganisms to obtain electrons from electrode to support denitrification processes. The microbial community divergence happens as a result of potential regulation. The dominant microorganisms at phylum level at different potentials are all belonged to Proteobacteria, Bacteroidota, and Actinobacteriota, with Chryseobacterium (31.35%), Unclassified Comamonadaceae (34.22%), and Azoarcus (16.53%) as the dominant genera. Additionally, electrode potential regulation shows a significant impact on the expression of functional gene and the reaction mechanism. Under -0.7 V, more napAB genes are expressed indicating that the denitrification is achieved by electron transfer through cytochromes, while, under -0.3 V and -0.5 V, denitrification process is achieved through electron transfer by the expression of narGHI genes.