Defect engineering has been attracted widespread attention for promoting the stability of the electrodes. However, accurately quantifying and defining the effect of defects is extremely difficult. Here, the Rietveld analysis with combined neutron powder diffraction (NPD) and X-ray powder diffraction (XRD) patterns reveal vanadium defect (Vd) clusters in the V2O3 lattice up to 5.7% in aqueous zinc-ion batteries (ZIBs), further confirmed by positron annihilation spectroscopy (PAS) and synchrotron-based X-ray analysis. Benefitting from the Vd clusters, the V2O3 cathode achieves excellent cycle life with 81% capacity retention at 5.0 A g-1 after 30,000 cycles that is the most superb stable cathode for aqueous ZIBs at this current density. Besides, the density functional theory (DFT) calculations strongly indicate that the Vd clusters not only provide permanent sites for Zn2+ anchormen to enhance the integrity of V2O3 after the first discharging process, but also make Zn2+ de/intercalation in complex oxide, contributing collectively and effectively reducing the strong electrostatic interaction between host multivalent ions, resulting in the remarkable storage performance of Zn2+. This work highlights accurately quantifying and identifying the significant effect of defects for designing cathodes with ultra-long cycle life in future intelligent devices.