Copper oxide (CuO) nanoparticles (NPs) doped by Ce4+ ions were prepared by hydrothermal method. FESEM images revealed doped CuO NPs were in spherical or subsphaeroidal shape. XRD patterns confirmed that Ce-doped CuO NPs exhibited the monoclinic structure at a dopant concentration less than 10%, and when the doping content was up to 15%, CeO2 phase formed. ICP analysis indicated that Ce4+ had a promoting effect on the release of Cu2+ from doped CuO NPs. The antibacterial activity of doped CuO NPs revealed that doped CuO NPs showed more effective bacterial resistance against Gram-positive Staphylococcus aureus (S. aureus) than Gram-negative Escherichia coli (E. coli) bacterium. Among them, 5% Ce-doped CuO NPs exhibited the best bactericidal effect at a very low concentration of 0.05 mg/mL. It was suggested that the binding of Cu2+ ions to the bacterial cell surface might play a major role in antibacterial activity.