In this study, a simple, rapid and sensitive luminol-CuO nanoparticles-H2O2 chemiluminescence (CL) method has been proposed for determination of glucose and cholesterol in plasma. Response surface methodology (RSM) based on Box-Behnken design (BBD) was used to optimize the most important operating variables (solution pH effect and the CL reagents concentration) of luminol CL system. In optimum conditions, it was found that CuO nanoparticles (NPs) could enhance the CL intensity and the method sensitivity towards evaluation of trace amount of glucose and cholesterol. Under the optimal conditions, there is a good linear relationship between the luminol-CuO NPs - H2O2 relative CL intensity and the concentration over the range of 1.2 × 10(-6)-1.0 × 10(-3) M (R (2) = 0.9991) for glucose and 2.5 × 10(-5)-CuO NPs7.17 × 10(-3) M (R (2) = 0.9968) for cholesterol and with a 3σ detection limit of 7.1 × 10(-7) and 6.4 × 10(-6) M, respectively. The relative standard deviations (RSD) < 3.3% were obtained.