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In the present study, inulin based nanocomposite viz., TiO2-MWCNT@Inulin was prepared by embedding Inulin (a biopolymer extracted from Allium sativum L.) with TiO2 and MWCNTs. The morphology of the prepared nanocomposite was characterized by High Resolution transmission electron microscopy (HRTEM). Cholesterol oxidase (ChOx) enzyme was then immobilized into the nanocomposite and the immobilization was examined by UV–vis and FT-IR spectral studies. The ChOx immobilized nanocomposite was integrated into carbon paste (CP) matrix to prepare the working electrode for the sensing of cholesterol. Electrochemical characterization of the modified CP/TiO2-MWCNT@Inulin/ChOx electrode was done by cyclic voltammetric (CV) and electrochemical impedance spectroscopic (EIS) studies. Differential pulse voltammetric (DPV) studies were carried out to determine the concentration of cholesterol at the interface of the newly fabricated electrode. The fabricated electrode demonstrated a linear range from 83 μM to 14.28 mM, low limit of detection (35 μM), good sensitivity (21.26 μA mM−1 cm-2), low Km (0.49 mM), high stability (120 days) and good selectivity. The presence of Inulin biopolymer played a vital role in attaching ChOx enzyme firmly to the nanocomposite thereby enhancing the stability and electron transfer efficiency of the electrode. The analysis of product that was formed within the electrochemical cell during the electrochemical oxidation of cholesterol was performed by using sodium nitroprusside. This resulted in a deep purple coloured solution which suggested the electrochemical conversion of cholesterol to cholestenone. The practical applicability of the fabricated electrode was also assessed by the determination of cholesterol in spiked blood serum and milk samples.