Abstract In this study, a novel non-enzymatic sensor based on copper oxide nanoparticles (CuO), polyaniline nanofibers (PANI) and murexide (Mu) modified glassy carbon electrode was developed and used for the detection of cholesterol. Copper oxide nanoparticles were deposited on the glassy carbon electrode through electrodeposition and electrochemical oxidation followed by electrodeposition of PANI-Mu composite. The as prepared CuO-PANI-Mu sensor was characterized using electrochemical, optical and morphological methods such as cyclic voltammetry (CV), impedance spectroscopy (EIS), linear sweep voltammetry (LSV), UV-visible and scanning electron microscopy (SEM). The elaborated composite matrix was used for cholesterol detection employing the impedance spectroscopy method. As a result, good analytical performances were obtained for cholesterol quantification with good stability and high sensitivity (5575 Ω/M) with a wide linear range from 0.5nM to 50mM.