Poly(o-toluidine) films were electrochemically synthesized on Pt electrodes at a constant potential (0.75 V versus Ag/AgCl) from a deoxygenated aqueous solution of 0.1M toluidine dissolved in 0.1M KCl. To form permselective polymeric film electrodes, poly(o-toluidine) films at different thicknesses were prepared by varying the amount of charge consumed during electrochemical polymerization. Then, experimental parameters (e.g., concentrations of monomer and electrolyte and pH of the phosphate buffer salt solution) affecting the polymeric film thickness were optimized. Permeation of the various electroactive and nonelectroactive species such as ascorbic acid, oxalic acid, hydrogen peroxide, lactose, sucrose, and urea through the optimized poly(o-toluidine)-coated electrodes was investigated using a chronoamperometric technique. From experimental results, it was found that a poly(o-toluidine)-coated electrode permitted the oxidation of hydrogen peroxide and prevented the permeation of the mentioned electroactive and nonelectroactive species. in other words, it was seen that this polymeric electrode responded to only hydrogen peroxide selectively. Thus, it has been claimed that a poly(o-toluidine)-coated Pt electrode can be used as a permselective polymeric membrane to overcome interference problems occurring in the hydrogen peroxide-based biosensor applications.