A simple, sensitive cupric oxide nanoparticles (CuO NPs) enhanced chemiluminescence (CL) method was developed for the measurement of β-lactam antibiotics, including amoxicillin and cefazolin sodium. The method was based on suppression of the CuO NPs-luminol-H2O2 CL reaction by β-lactam antibiotics. Experimental parameters that influenced the inhibitory effect of the antibiotic drugs on the CL system, such as NaOH (mol/L), luminol (µmol/L), H2O2 (mol/L) and CuO NPs (mg/L) concentrations, were optimized. Calibration graphs were linear and had dynamic ranges of 1.0 × 10(-6) to 8.0 × 10(-6) mol/L and 3.0 × 10(-5) to 5.0 × 10(-3) mol/L for amoxicillin and cefazolin sodium, respectively, with corresponding detection limits of 7.9 × 10(-7) mol/L and 1.8 × 10(-5) mol/L. The relative standard deviations of five replicate measurements of 5.0 × 10(-6) amoxicillin and 5 × 10(-4) cefazolin sodium were 5.43 and 5.01%, respectively. The synthesized CuO NPs were characterized by X-ray diffraction (XRD) and transmission electronmicroscopy (TEM). The developed approach was exploited successfully to measure antibiotics in pharmaceutical preparations.