An electrochemical biosensor was prepared by immobilizing cholesterol oxidase (ChOx) on a nanocomposite consisting of amine functionalized multi-walled carbon nanotubes and a room temperature ionic-liquid (1-butyl-3-methylimidazolium tetrafluoroborate). The biosensor was examined for cholesterol quantification in clinical serum samples. ChOx on the modified electrode exhibited a couple of quasi-reversible redox peaks corresponding to the direct electron transfer of FAD/FADH₂ buried in the enzyme structure. A formal potential of −400 mV was obtained for the immobilized enzyme in phosphate buffer solution (0.1 M, pH 7.4). Under the optimized experimental conditions, the biosensor exhibited a linear response towards cholesterol concentration from 26 nM to 3.4 μM, with a sensitivity of 540 μA mM⁻¹and a detection limit of 12 nM.