Efficient and robust non-platinum-group metal electrocatalysts for O2 reduction are a prerequisite for practical high-performance fuel cells and metal-air batteries. Herein, we reported an integrated principle of gradient electrospinning and controllable pyrolysis to fabricate various Co-doped Ni3V2O8 nanofibers with high oxygen reduction reaction (ORR) activity. The representative Co1.3Ni1.7V2O8 nanofibers showed outstanding ORR performance in an alkaline solution with a half-wave potential (E1/2) of 0.874 V vs RHE, along with high long-term stability. Furthermore, the introduction of Co could effectively restrain the growth of nanoparticles and change the electronic structure of Ni3V2O8. Control experiments and theoretical calculations demonstrated that upon Co-doping, the hybridization between the 3d orbital for both Co and Ni guaranteed the stable adsorption interaction with O2 over Ni and Co metal centers. Meanwhile, the weakened binding ability of Ni3V2O8 to OH* reduced the ORR free energy. Overall, the synergistic effect of Co and Ni metal cations essentially reflected the origin of ORR activity on the Co-doped Ni3V2O8 nanofibers. This work offers new insights and practical guidance for designing highly active ORR catalysts for electrochemical clean energy conversion and storage.