Oxygen vacancy (Vö) is important in the modification of electrode for rechargeable batteries. However, due to the scarcity of suitable preparation strategy with controllable Vö incorporation, the impact of Vö concentration on the electrochemical performances remains unclear. Thus, in this work, Vö-V₂O₅-PEDOT (VöVP) with tunable Vö concentration is achieved via a spontaneous polymerization strategy, with the capability of mass-production. The introduction of poly(2,3-dihydrothieno-1,4-dioxin) (PEDOT) not only leads to the formation of Vö in V₂O₅, but it also results in a larger interlayer spacing. The as-prepared Vö-V₂O₅-PEDOT-20.3% with Vö concentration of 20.3% (denoted as VöVP-20) is able to exhibit high capacity of 449 mAh·g⁻¹ at current density of 0.2 A·g⁻¹, with excellent cyclic performance of 94.3% after 6,000 cycles. It is shown in the theoretical calculations that excessive Vö in V₂O₅ will lead to an increase in the band gap, which inhibits the electrochemical kinetics and charge conductivity. This is further demonstrated in the experimental results as the electrochemical performance starts to decline when Vö concentration increases beyond 20.3%. Thus, based on this work, scalable fabrication of high-performance electrode with tunable Vö concentration can be achieved with the proposed strategy.