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Cholesterol biosensor based on direct electron of cholesterol oxidase(ChOx) covalently funtionalized on multi-wall carbon nanotubes(MWNTs) modified screen printed electrode (SPE) was studied. The MWNTs/ChOx/SPE has been characterized using electrochemical method including cyclic voltammetry and amperometry. Carbon Nanotubes can improve the direct electron transfer of ChOx and the electrode surface. This biosensor has good electrochemical behaviour and stability. In addition, the interferences compound in real sample such as ascorbic acid (AA), uric acid (UA), 4-acetamidophenol (AP), etc. did not cause any interference of this biosensor due to the use of a low potential from direct electron transfer of ChOx which is -0.4 V.