We introduced single-charged molecules into layer-by-layer film of polymers by mixing polyelectrolyte and single-charged molecules with an appropriate ratio to form polymer complex and then deposition in alternation with counter-polyelectrolyte. By immersing the layer-by-layer film into aqueous solution of electrolytes, the single-charged molecules could be released from the layer-by-layer film. An interesting finding is the releasing of the single-charged molecules gave the multilayer film the permselectivity. For example, cations would be trapped and anions be repelled if positive-charged molecules were used. Multilayer films able to trap either cations or anions could be fabricated, depending on the nature of single-charged template molecules used. Thus, the ion permselectivity of multilayer film was enhanced. Moreover, the permeability of the film was also dramatically increased, suggesting the potential application of layer-by-layer films as highly efficient ion-permselective membranes.