By means of electrochemical polymerization, polybenzidine-modified electrodes were prepared in an aqueous monomer solution at a potential of 0.7 V versus Ag/AgCl. The permselective character of the polybenzidine electrode prepared in a one-step procedure was examined for electroactive (ascorbic acid, oxalic acid, and hydrogen peroxide) and nonelectroactive (lactose, sucrose, and urea) species. Influence of the various parameters on the permselective properties of the polybenzidine membrane was systematically investigated and the optimal values for these parameters were determined. It has been found that polybenzidine membrane showed selective permeation for hydrogen peroxide while blocking the permeation of electroactive and nonelectroactive interferents through film. In brief, it is claimed that this polybenzidine film can be used as a coating material to prevent interferences in electrochemical biosensor applications.