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Much attention has been given to aqueous zinc-ion batteries (ZIBs) due to their features of inherent safety, environmental compatibility, low cost, and fantastic energy density. Nevertheless, chemical corrosion and dendrite growth occurring on Zn anodes during the charge–discharge process, which often cause surface passivation and short circuit of cells, seriously hindering the development of ZIBs. To solve these problems, a Cu(II) and polydopamine co-modified glass fiber (Cu(II)-PDA/GF) is designed as separator. On one hand, the modification of PDA enhances ionic conductivity and the water absorbing capability of a glass fiber separator due to the presence of functional groups. On the other hand, the pre-deposition of Cu on Zn anodes enables the uniform nucleation of Zn during the initial deposition process. Due to the synergistic effect, reversible zinc plating/striping is achieved in symmetric cells, which display a long lifecycle of over 1800 h at the current density of 1 mA cm⁻² and with a fixed capacity of 1 mAh cm⁻². Moreover, the assembled Zn//V₂O₅ cells using the Cu(II)-PDA/GF separator also demonstrate improved capacity retention. This study provides a simple and effective separator modification strategy for high-performance and reliable ZIBs, which are conducive to other metal-based energy storage devices.