Precise identification of cholesterol levels is crucial for the early diagnosis of cardiovascular risk factors. This paper presents a novel approach for cholesterol detection that circumvents the reliance on enzymatic processes. Leveraging the unique properties of advanced materials and electrochemical principles, our non-enzymatic approach demonstrates enhanced sensitivity, specificity, and limit of detection in cholesterol analysis. A non-enzymatic electrochemical biosensor for Cholesterol, employing a nanohybrid comprising Cu2O nanoparticles decorated with MoS2, is presented. The cyclic voltammetry (CV), differential pulse voltammetry (DPV), and amperometry techniques were employed to investigate the electrochemical behaviour of the glassy carbon electrode modified with the Cu2O/MoS2 nanohybrid. The modified electrode exhibited an excellent sensitivity of 111.74 μA μM−1 cm−2 through the CV method and showcased a low detection limit of 2.18 μM and an expansive linear range spanning 0.1–180 μM when employing the DPV method. The electrode also showed good selectivity to various interfering components in 0.1 M NaOH and a satisfied stability of about 15 days at room temperature. The study demonstrates the potential for broader applications in clinical diagnostics and monitoring cardiovascular health, paving the way for a paradigm shift in cholesterol detection methodologies and offering a more efficient and cost-effective alternative to traditional enzymatic assays.