A simple and high sensitive cholesterol amperometric biosensor, which is based on in situ electropolymerization of multi-walled carbon nanotube-polyaniline (MWCNT-PANI) nanocomposite and electrodeposition of platinum nanoparticle (nano-Pt) films onto the glassy carbon electrode surface for cholesterol oxidase immobilization, was constructed in this study. The preparation process of the modified electrode was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, scanning electron microscopy, and chronoamperometry. Because of the synergistic electrocatalytic activity between MWCNT-PANI nanocomposites and nano-Pt, the cholesterol biosensor exhibited an excellent performance with a linear range of 2.0-510.0 µM, a detection limit of 0.8 µM (signal-to-noise ratio = 3), a high sensitivity of 109.9 µA mM , and a short response time within 5 Sec. Moreover, the reproducibility, stability, and selectivity of the biosensor were also investigated.