The preparation of single atom (SA) Fe-N-C catalysts always results in the formation of an oxygen(O)-containing moieties. The effect of the oxygen-containing part on the oxygen reduction reaction (ORR) conducted with Fe-N-C is often overlooked, and the mechanism of the contribution of the oxygen-doping structure remains ambiguous. Therefore, guided by density functional theory (DFT) calculations, the adjacent C-O-C structure promotes the charge separation of the active center of FeN₄ and reduces the adsorption of O-containing intermediates, which significantly increase the ORR kinetics. To experimentally verify such principle, we design the SA FeN₄ catalysts (FeN₄-700/900) incorporating in carbon substrates derived from O-rich biomass. The FeN₄-700/900 catalyst displays enhanced ORR activity under acidic (0.78 V) and alkaline (0.904 V) conditions over FeN₄-700/700 and FeN₄-900/900 catalysts, while exhibiting excellent Zn-air battery performance. This work opens an avenue to explore the modulation of the active site and the practical application of Fe-N-C catalysts.