The development and evaluation of a novel separator material for aqueous zinc-ion batteries (ZIBs) are discussed, which are promising for energy storage due to their use of abundant zinc metal, eco-friendly electrolytes, and high safety. The main challenges in ZIBs are the formation of zinc dendrites and the hydrogen evolution reaction (HER), which can lead to short circuits and reduced battery life. To address these issues, the authors have functionalized chitin separators through a process involving alkaline treatment and mechanical grinding, resulting in nano-chitin fibers with varying degrees of deacetylation (D-x-ChNF). The D-4-ChNF separator, in particular, has been shown to have uniformly distributed nanochannels, high mechanical strength, and excellent electrochemical stability. It also exhibits a strong affinity for aqueous ZnSO4 electrolytes and enhances the electrochemical reversibility of zinc through increased coordination with Zn2+ ions. This leads to a significant improvement in the battery’s cycle life, with the D-4-ChNF separator outperforming traditional glass fiber (GF) separators by more than six times in cycle life at 5 mA cm−2 and 5 mAh cm−2. The D-4-ChNF separator also demonstrates superior rate performance and long-term cycling stability, with capacity retention rates of 90.46% and 96.68% after 1000 cycles at 5 A g−1 and 10 A g−1, respectively. The separator’s ability to suppress dendrite growth and improve the uniformity of zinc deposition is attributed to its ability to reduce nucleation overpotential and promote uniform zinc deposition along the (002) crystal plane. The D-4-ChNF separator, with its low cost and high stability, offers a promising solution for enhancing the performance and practical application of ZIBs, providing new insights into the development of efficient and sustainable energy storage technologies.