An atomically dispersed Fe-N-C catalyst has been synthesized and enables high power-out performance in microbial fuel cells (MFCs). The influence of Fe doping on the electronic properties of N-doped carbon was investigated, proving that single-atomic Fe sites embedded into N-doped carbon play a significant role in enhancing oxygen reduction reaction (ORR) performance in challenging neutral electrolyte. Density functional theory (DFT) studies indicate that a lower energy barrier for the limiting-potential step (*OH desorption) on Fe-N4 sites is favorable for the ORR process. This work offers new insights into the nature of Fe-N4 sites for the construction of highly active electrocatalysts for use in diverse energy conversion applications.