A dithiobissuccinimidyl propionate (DTSP) self-assembled monolayer (SAM) prepared onto a gold (Au) surface has been utilized for covalent immobilization of cholesterol oxidase (ChOx) and cholesterol esterase (ChEt). These ChOx-ChEt/DTSP/Au bio-electrodes have been characterized using electrochemical impedance and cyclic voltammetric (CV) techniques, respectively. Differential pulse voltammetry (DPV) has been used for enzymatic assay of immobilized ChOx and ChEt onto the DTSP modified gold surface as a function of cholesterol oleate concentration. The response measurement conducted on ChOx-ChEt/DTSP/Au bio-electrode reveal the value of Michaelis-Menten constant (Km) as 0.95 mM suggesting enhanced affinity of enzymes (ChOx and ChEt). The ChOx-ChEt/DTSP/Au bio-electrodes show linearity in range of 50 to 400 mg dl(-1) of cholesterol oleate and the shelf-life of more than 50 days when stored at 4 degrees C. This biosensing electrode shows correlation coefficient of 0.9973 and standard deviation of regression as 0.859 microA.