A novel fabrication approach of a highly sensitive amperometric glucose biosensor based on a single ZnO nanofiber (ZONF) is presented. Nanofibers (NFs) of poly(vinyl pyrrolidone)/zinc acetate composite have been synthesized by electrospinning technique. By high-temperature calcinations of the above precursor fibers, ZONFs with diameters in the range of 350-195 nm have been successfully obtained. A single NF on a gold electrode is functionalized with glucose oxidase (GOx) by physical adsorption. Electrochemical measurements of the biosensor revealed a high and reproducible sensitivity of 70.2 mu A cm(-2) mM(-1) within a response time of less than 4 s. The biosensor also showed a linear range from 0.25 to 19 mM with a low limit of detection (LOD) of 1 mu M. Furthermore, it has been revealed that the biosensor exhibits a good anti-interference ability and favorable stability over relatively long-term storage (more than 4 months). All these results strongly suggest that a single ZONF can provide a new platform for biosensor design and other biological applications.