In this study, a CNT fiber flexible electrode grafted with CuO nanoparticles (CuO NPs) and polyaniline (PANI) was developed and applied to a nonenzymatic electrochemical sensor for H2O2 detection. CuO NPs/PANI/CNT fiber electrode was fabricated through the synthesis and deposition of PANI and CuO NPs on the CNT fiber surface using an electrochemical method. Surface morphology and elemental composition of the CuO NPs/PANI/CNT fiber electrode were characterized by scanning electron microscope with energy dispersive X-ray spectrometry. And its electrochemical characteristics were investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA). Compared with a bare CNT fiber as a control group, the CuO NPs/PANI/CNT fiber electrode showed a 4.78-fold increase in effective surface area and a 8.33-fold decrease in electron transfer resistance, which leads to excellent electrochemical properties such as a good electrical conductivity and an efficient electron transfer. These improved characteristics were due to the synergistic effect through the grafting of CNT fiber, PANI and CuO NPs. As a result, this electrode enhanced the H2O2 sensing performance.