Cephalexin (Ceph) is a semi-synthetic antibiotic of the cephalosporins group, which can cure a number of bacterial infections. It kills gram-positive and some gram-negative bacteria by interrupting the growth of the wall of bacterial cell. It is an effective broad spectrum antibiotic, which have a structure like to that of the penicillin and is used for patients allergic to penicillin [1]. The chemical structure of Ceph was shown in Fig.1. Generally, medicines can pollute water sources because of the discharge of chemical industry and hospital wastes into the environment. Most of these chemical compounds may persistent in the environmental waters for a long period of time and can cause harmful effect on aquatic animal organisms and human health. Thus, measurement of pharmaceuticals in water samples is very important. Furthermore, determination of Ceph in pharmaceutical products is very essential process in the quality control of the pharmaceutical industries [2]. In this work, a new and sensitive CuO nanoparticles (CuO NPs)–catalysed H2O2–HCO3− system was developed for determination of Ceph. The suggested method was based on inhibitory effect of Ceph on the CL system. The experimental parameters affecting the CL intensity including concentrations of NaHCO3, CuO NPs and H2O2 were optimized. Under the optimum condition, the calibration curve was plotted for Ceph. It was linear in the concentration of the range of 5.0 × 10−5 –1.0× 10−3 mol L−1, with detection limit of 1.2 × 10−5 mol L−1. In addition, the CuO NPs were synthesized by using quick-precipitation method and characterized by employing X-ray diffraction (XRD) and scanning electron microscopy (SEM) techniques. The method was successfully exploited for measurement of Ceph in the pharmaceutical preparations and tap water.