Microchip capillary electrophoresis (MCE) has significant impact on diagnostic testing. One of the most importance in clinical analysis test is the determination of cholesterol level in blood, because its increase is associated with coronary heart disease which is a major cause of death world over. Poly- dimethylsiloxane (PDMS) was material used to make a MCE for determination of cholesterol. Hydrogen peroxide was generated from the oxidation of cholesterol with cholesterol oxidase (ChOx) and was detected electrochemically at a downstream gold (Au) wire electrode by amperometry. Various parameters, such as the detection potential, concentration of running buffer, pH of running buffer, separation voltage, injection time, and concentration of ChOx, were studied. The migration time of cholesterol is less than 100 s and the calibration curve is linear from 50 mg·dL-1 to 250 mg·dL-1 with the coefficient of determination (R2) of 0.9955. Therefore, this proposed assay is very rapid and sensitive for the detection of cholesterol.