A novel poly(styrene--epsilon-caprolactone)/multiwalled carbon nanotubes/cholesterol oxidase film-coated glassy carbon electrode was designed for cholesterol detection by square wave voltammetry (SWV). The biosensor responded to cholesterol with a measurement concentration range between 1 and 130 mu M, a relative standard deviation of only 0.095% and accuracy of 100.42% +/- 2.85 with the SWV technique in the potential range from -0.6 to +0.6 V. The limit of detection was calculated to be 0.63 mu M. The biosensor was preserved 91 and 84% of its initial response at the end of the 9st and 25st days, respectively. Human serum from human male AB plasma was analyzed without pretreatment except for dilution to investigate the performance of the biosensor in a complex medium.