We report a novel composite electrode made of chitosan-SiO 2 -multiwall carbon nanotube (CHIT-SiO 2 -MWNT) composite coated on the indium-tin oxide (ITO) glass substrate. Cholesterol oxidase (ChOx) was covalently immobilized on the CHIT-SiO 2 -MWNT/ITO electrode that resulted in a ChOx/CHIT-SiO 2 -MWNT/ITO cholesterolactive bioelectrode. The CHIT-SiO 2 -MWNT/ITO and ChOx/CHIT-SiO 2 -MWNT/ITO electrodes were characterized with Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The influence of various parameters was investigated, including the applied potential, pH of the medium, and the concentration of the enzyme on the performance of the biosensor. The cholesterol bioelectrode exhibited a sensitivity of 3.4 nA/ mgdL ร1 with a response time of five seconds. The biosensor using ChOx/CHIT-SiO 2 -MWNT/ITO as the working electrode retained its original response after being stored for six months. The biosensor using ChOx/CHIT-SiO 2 -MWNT/ITO as the working electrode showed a linear current response to the cholesterol concentration in the range of 50 -650 mg/dL.