A glassy carbon and indium tin oxide (ITO) electrodes have been modified with the nano TiO2–Au–KI film by the adsorption of TiO2 nanoparticles on the electrodes followed with the electrochemical depositions of nano Au and KI film. Further the nano TiO2–Au–KI film modified ITO was examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. From the microscopic results, the adsorbed nano TiO2 particles size were found in the range of 70–100 nm. Here the electrochemical depositions of nano Au were formed as a flower shape were in the size range of 230 nm to 1 μm. Next the electrochemical behavior of nano TiO2–Au–KI film has been examined in different pH solutions. The nano TiO2–Au–KI film modified glassy carbon electrode (GCE) showed good electrocatalytic activity towards the oxidation of H2O2 in phosphate buffer solutions (pH 7.0). The linear range of detection for H2O2 oxidation using nano TiO2–Au–KI film was found as 1 × 10−5 to 1 × 10−4 M and 1 × 10−9 to 1 × 10−7 M in CV and differential pulse voltammetry (DPV) techniques. The practical applications of nano TiO2–Au–KI film was evaluated by analyzing the real samples such as antiseptic and contact lens cleaner solutions containing H2O2.