A novel amperometric biosensor was fabricated based on the immobilization of cholesterol oxidase (ChOx) into a cross-linked matrix of chitosan (Chi)-room-temperature ionic liquid (IL) (1-butyl-3-methylimidazolium tetrafluoroborate). Initially, the surface of bare electrode (indium tin oxide coated glass) was modified with the electrodeposition of Au particles onto thiol (-SH) functionalized multi-walled carbon nanotubes (MWNTs). The biosensor electrode is designated as MWNT(SH)-Au/Chi-IL/ChOx. Scanning electron microscopy image of MWNT(SH)-Au/Chi-IL/ChOx reveals that Chi-IL exists as the interconnected wires covering the Au particles on the surface of MWNT(SH)-Au. Cyclic voltammetry and chronoamperometry were used for the electrochemical determination of cholesterol at the biosensor electrode, MWNT(SH)-Au/Chi-IL/ChOx. The presence of Au particles in the matrix of CNTs provides an environment for the enhanced electrocatalytic activities. The MWNT(SH)-Au/Chi-IL/ChOx biosensor exhibited a linear response to cholesterol in the concentration range of 0.5-5mM with a correlation coefficient of 0.998, good sensitivity (200 microAM(-1)), a low response time ( approximately 7s), repeatability (R.S.D value of 1.9%) and long term stability (20 days with a decrease of 5% response). The synergistic influence of MWNT(SH), Au particles, Chi and IL contributes to the excellent performance for the biosensor.