Porous membranes with different charge on the surface and internal pore walls are prepared via the solvent-responsive layer-by-layer (SR-LBL) method. The effect of charge on the transport properties of different ions through the membranes is investigated in detail. The charge property of prepared membranes is tuned by assembling different charged polyelectrolytes (PEs) on the pore walls and the surface of the porous membranes. The results show that in a vanadium flow battery (VFB), the PE layers assembled on the surfaces (including pore walls) are capable to construct excellent ion transport channels to increase proton conductivity and to tune the ion selectivity via Donnan exclusion effect. Compared with the porous membrane with negative charges (7 bilayers), a VFB single cell assembled with a positively charged membrane (7.5 bilayers) yields a higher coulombic efficiency (98%). The water and ion transfer behavior exhibits a similar tendency. In the negative half-cell, the amount of V3+ gradually increases as cycles proceed and the amount of V2+ stays at a low and stable level. In the positive half-cell, the amount of VO2+ decreases; while VO2 + is accumulated. The imbalance of vanadium ions at both sides induces the discharge capacity fade.