Single-atom catalysts have recently emerged as a promising replacement for noble metal catalysts in the oxygen reduction reaction (ORR) on the cathode side of fuel cells and metal-air batteries. However, regulating the precise coordination environment of central atoms remains challenging. In particular, constructing co-coordination active site with N and O atom is rarely investigated. Here, an unreported pre-oxidation strategy is proposed to establish the Fe-O bond for the synthesis of the Fe-N4O1 single-atom structure. The obtained SA-FeN4O1-C catalyst exhibits remarkable ORR performance, possessing a half-wave potential 30 mV higher than that of the commercial Pt/C and a kinetic current density 7.4 times that of Pt/C at 0.8 V (vs. RHE). More significantly, when employed as an air cathode, the overpotential is 340 mV lower than that of Pt/C when operating at a high current density of 350 mA cm−2 in a GDE half-cell, and a maximum power density of 205.8 mW cm−2 and a high energy density of 1024.9 w h kg−1 can be achieved in a zinc-air battery. According to the DFT, axial oxygen coordination can lower the energy barrier for ORR by driving the d-band center of the Fe atom to shift away from the Fermi level and generate a moderate oxygen adsorption energy. Importantly, the essence of the formation of the Fe-N4O1 structure and single-atomic active sites was revealed. This work opens up an innovative strategy for constructing single-atom sites with axial oxygen coordination and provides valuable insight into the synthesis of atomically dispersed materials.