In order to improve the denitrification efficiency of low carbon to nitrogen ratio (C/N) wastewater, we conducted continuous flow experiments of weakly electrically stimulated denitrification using a direct current output voltage. The results showed that the best denitrification was achieved at a voltage of 0.2 V. The removal of nitrate and total nitrogen was increased by 20% and the production of intermediate greenhouse gas (N2O) was reduced by 62.6%. We explored the specific pathways involved in the weak electrical stimulated denitrification using enzyme activity as a cut-off point. The enzyme activity analysis and 3D fluorescence spectroscopy revealed that nitrate reductase (NAR) and nitrite reductase (NIR) activities were significantly enhanced by weak electrical stimulation, and the aromatic protein content in extracellular polymeric substances (EPS) increased, accelerating electron transfer and promoting the conversion of loosely bound EPS (LB) to tightly bound EPS (TB). The accelerated electron transfer further increased enzyme activity and the metabolic rate of microorganisms. This study indicates that weak electrical stimulation could improve activities of biological enzymes to enhance denitrification efficiency.