Vanadium redox flow batteries (VRFB) have been widely used as grid-scale energy storage systems because of their many advantages, such as moderate operating conditions, large-scale capacity, long cycle life, and relative safety. Despite these advantages, VRFBs suffer from the critical problem of electrolyte contamination due to vanadium ion crossover through the separator (Nafion 212), with consequent capacity decay during cycling. In this study, to reduce the crossover of vanadium ions, Nafion 212 was modified with polydiallyldimethylammonium chloride (PDDA) and cellulose nanocrystals (CNC) via a layer-by-layer process. Nafion-[PDDA/CNC] n was stacked to a thickness of 8.92 nm per layer (n = 1). As the stacking number, n, increased, the area resistance increased exponentially, indicative of degradation of the cell performance due to an increase in the mass transfer resistance. Nevertheless, the resistance against self-discharge, which is measured in terms of the change in the open circuit voltage (OCV), was dramatically enhanced as a function of the number of stacking layers; the OCV was maintained for up to 150 h due to restriction of vanadium ion crossover. Thus, the optimum stacking number for maximizing the cell performance is twenty (Nafion-[PDDA/CNC] 20 ) based on the trade-off effect between degradation of the membrane permeability and increased resistance.