Functional carbon nanotubes (CNT) have attracted much attention for analytical and biomedical applications. This paper describes the fabrication of a cholesterol oxidase (ChOx) immobilised polyaniline (PANI)/CNT composite electrode for the amperometric detection of cholesterol. The prepared ChOx/PANI/CNT/Au bioelectrode bound ChOx via the available functionalties of PANI (-NH2) and CNT (-COOH). Moreover, the CNT creates a network inside the matrix that strengthens the mechanical property of the bioelectrode. The multifunctional matrix is presumed to provide a 3D-mesoporous surface, which substantially enhances enzyme activity. The linear range of the biosensor for cholesterol oleate was 30-280 μM with a response time of 10 sec.