To prevent bacterial contamination and enhance wear-resistance of titanium-based tools and implants, it is crucial to develop functional coatings with inherent antimicrobial property and high surface hardness. In this study, the micro-arc oxidation technique was employed to prepare ceramic oxide films with antibacterial properties on titanium metal surfaces by incorporating trace amounts of copper. It was found that the phytic acid (C6H18O24P6) used as the chelating agent in the Cu-containing electrolyte can not be recycled and the adding of alternative chelating agent calcium gluconate (C12H22O14Ca) can significantly promote the cyclic utilization. The oxide film prepared by using C12H22O14Ca as the chelating agent exhibits a typical volcanic micro-arc oxidation morphology with uniform copper distribution, high hardness and excellent substrate adhesion. Most importantly, in vitro antibacterial tests demonstrate the outstanding antibacterial performance of Cu-doped TiO2 film with an antibacterial rate of up to 99.9% against Escherichia coli and Staphylococcus aureus.