Aqueous zinc metal batteries (AZMBs) hold promises for extensive energy storage applications due to the water-based electrolyte’s affordability and environmental friendliness. One integral component of the battery is separator, which serves as an insulator for electrons and allows ions movement between the positive (cathode) and negative (anode) electrodes. Glass fiber is currently the most extensively used separator in AZMBs research. However, it is excessively thick (∼150 µm), which reduces the volumetric energy density of the battery in practical application. Therefore, a thin separator with robust mechanical properties is desired to advance the technology. In this work, we developed cellulose-based separator via solution casting process, resulting in a separator with ∼30 µm thickness. The separator is made from cellulose nanocrystal (CNCs) with sodium as the counter ion and TEMPO-oxidized cellulose nanofibrils (TCNF) with carboxylate surface charge. The functional groups present in the cellulose such as hydroxyl and carboxylate groups give advantage for the zinc ion transportation between the electrodes. Electrochemical investigation has been carried out to evaluate the separator performance in AZMB. Battery testing in an asymmetric cell with zinc metal as the counter electrode, titanium as the working electrode, and the fabricated cellulose separator in between the two electrodes demonstrate hat the separator can work for Zn plating/stripping at practical current density and areal capacity of 10 mA cm-2 and 5 mAh cm-2, respectively. Furthermore, to acquire a real-time understanding of Zn plating/stripping process with the prepared cellulose separator, synchrotron X-ray microtomography (SXTM) will be employed. The SXTM image data provides both qualitative and quantitative information that can be utilized to assess the role of our prepared cellulose separator in the Zn plating/stripping in AZMB.