The flavin adenine dinucleotide (FAD) modified zinc oxide films have been prepared using repeated cyclic voltammetry to investigate both the deposition process and the films’ electrocatalytic properties. This paper describes the successful loading of electrochemically active molecules into ZnO by electrochemical method. The cyclic voltammograms recorded the direct deposition of the FAD/zinc oxide films over different scanning potential ranges from the mixed aqueous Zn2+ ions and FAD. In addition to the cyclic voltammetry, an electrochemical quartz crystal microbalance, UV–visible absorption spectroscopy, and the stopped-flow method were used to study the growth mechanism and their properties of the FAD/zinc oxide films. The results showed that the Zn2+ ions and FAD reacted by an electrochemical process to form a self-assembly FAD modified zinc oxide film. The FAD/zinc oxide films exhibited a single redox couple that included both the electron and proton transfer, with a formal potential that demonstrated a proton effect in acidic and basic solutions. The electrocatalytic reduction of NAD+ employing the FAD/zinc oxide films was investigated by cyclic voltammetry and UV–visible absorption spectroscopy methods. The electrocatalytic reduction of S 4 O 6 2 - , SO 5 2 - , S 2 O 8 2 - , ClO 3 - , BrO 3 - , and IO 3 - ions using a FAD/zinc oxide film occurred in neutral aqueous solutions.