Electrospun carbon nanofibers (CNFs) decorated with copper oxide nanoparticles were successfully synthesized using a one step and modified hydroxyl ion assisted alcohol reduction method. The X-ray diffraction pattern reveals the presence of (-111), (111) and (-202) crystal planes of monoclinic copper oxide (CuO). The CuO coated CNF air annealed at 250 degrees C exhibited an electrical conductivity of 251 S m (1). The CuO coated CNFs were employed as a novel flexible matrix for a cholesterol biosensor. The biosensor exhibited a high sensitivity of 75 mu A mM(-1) cm(-2) over the linear range (0.12 mM to 12.93 mM) of cholesterol. This coating method offers easy and inexpensive processing with a flexible and robust supporting structure for biosensing applications.