In this study, CuO was synthesized using a facile precipitation method and was characterized using various techniques for the efficient activation of periodate (PI). Compared to systems consisting of PI alone (0.5 mM) and CuO alone (0.5 g/L), the CuO (0.5 g/L)/PI (0.5 mM) process exhibited improved degradation performance for ciprofloxacin (CIP), a widely used refractory antibacterial drug, with 98% of this pollutant (20 mg/L) removed in a reaction time of 30 min. Both radical and nonradical degradation pathways were identified to play a significant role in the developed CuO/PI system, where the nonradical pathway (holes and electrons) played the most important role in the degradation of CIP. Under both acidic (pH = 3) and near-neutral (pH = 6) conditions, the system showed an improved efficiency for the degradation of CIP in comparison with alkaline conditions (pH = 10). This was attributed to a more facile adsorption of the PI ions onto the surface of the CuO nanoparticles, induced by the electrostatic attraction between the PI ions and the CuO nanomaterial. Analysis of the degraded samples with ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC Q-TOF MS) demonstrated that cleavage of the piperazine ring and defluorination were the primary degradation pathways of CIP under this oxidation process. The CuO/PI system also showed an excellent removal efficiency for other refractory organic contaminants, such as sulfamethoxazole (SMX) and methylene blue (MB), in various water matrices, including real wastewater. Moreover, respiration tests revealed that the toxicity of the effluents was reduced after the treatment process. In conclusion, this research presents a novel, promising periodate activation technique for the removal of refractory organic contaminants in wastewater treatment.