A polymelatonin film was prepared using repetitive cyclic voltammetry. Then, FAD (flavin adenine dinucleotide)/toluidine blue O (TBO)/polymelatonin hybrid films were prepared by a chemical of FAD and TBO on polymelatonin films. Electrochemical quartz crystal microbalance, cyclic voltammetry, and scanning electron microscopy were used to study the growth mechanism of the hybrid FAD/TBO/polymelatonin films. The hybrid FAD/TBO/polymelatonin films exhibited three redox couples with formal potentials at -0.09, 0.19, and 0.39 V, respectively. The electrocatalytic property of the O-2 reduction was studied by using the hybrid films in the absence and presence of hemoglobin or myoglobin. The electrocatalytic oxygen reduction current has been developed from the cathodic peak of the FAD redox couple. The electrocatalytic process involved an interaction of hemoglobin, (myoglobin or cytochrome C), and FAD in hybrid film to increase the electrocatalytic reduction current. The electrocatalytic reduction of O-2 using the hybrid films was investigated by cyclic voltammetry and rotating ring-disk electrode methods. The electrocatalytic oxidation of dopamine and ascorbic acid using the hybrid films were also investigated. (c) 2006 The Electrochemical Society.