To confirm the viability of a porous polyethylene (PE) separator for using in vanadium redox flow batteries (VRFBs), we conduct a comparative electroanalytical and chemical stability studies of the PE separator and Nafion 212 membrane. We characterize the physicochemical properties of the separator, such as water uptake, dimensional change, and ion conductivity, and analyze its structural and compositional features using thermogravimetric analysis, scanning electron microscopy, and X-ray photoelectron spectroscopy. We also test the chemical stability of the separator against highly oxidative V5+ ions and the cell performance of VRFB using PE separator to assess practical applicability. In chemical stability, the change of VO2 + ion to VO2+ ion is considerably lowered by the PE separator (0.01 mmol/L) than the Nafion 212 (0.27 mmol/L), which indicates that the PE separator possesses higher chemical stability. The energy efficiency of the VRFB with the PE separator is lower than that obtained with Nafion 212. However, the chemical stability of PE separator is 27 times higher than that of Nafion 212, indicating that its use will promote the long-term operation of the VRFB system. Hence, the PE separator can be considered a cost-effective option for VRFB operation, with appropriate modifications to its thickness, surface properties and pore structure.