Nitrate (NO3−) in wastewater is a rising global threat to ecological and health safety. A sufficient carbon source, as the electron donor, is essential in the conventional biological denitrification process. It is not appropriate to add extra carbon sources into specific water bodies in terms of material cost and secondary pollution. Thus, innovative NO3− removal technologies that are independent of carbon sources, are urgently needed. This study constructed sediment microbial fuel cells (SMFCs) for aerobic denitrification in low-organic matter wastewater and explored the key factors affecting denitrification efficiencies. The SMFC treatments removed 72–91% NO3− through two main denitrifying stages which were driven by carbon sources (COD) and generated electrons, respectively. After COD was fully consumed, denitrification efficiencies were enhanced in SMFC treatments by 24–47% using the generated electrons within 3 days. In this stage, the NO3− removal efficiencies were positively correlated with external current intensities (p < 0.05). The improved denitrification efficiencies were attributed to two enriched phyla in the SMFC cathode. The dominant genera also demonstrated the heterotrophic denitrifying capacity of the SMFC biocathode. Furthermore, electrical characteristics could be used to monitor or regulate the denitrification process in the SMFC system. In conclusion, this study presents an innovative treatment strategy that is economical and eco-friendly compared with conventional physicochemical methods.