In this study, a simple, novel method of preparing glucose amperometric biosensors is reported. This biosensor is based on the quantitative measurement of an intermediate product, hydrogen peroxide (H2O2), which in turn is oxidized by Prussian blue film in composite nanofibers. The biocomposite is composed of Prussian blue, chitosan, and polyvinyl alcohol fabricated by electrodeposition and subsequent electrospinning in enzyme-friendly conditions. The resulting biocomposite nanofibers with porous structures and good biocompatibility sustained the stability of the Prussian blue film and the efficient enzyme immobilization without additional cross-linking agents. The stability of the Prussian blue film at neutral and weak alkalescent solutions was increased after the modification. Furthermore, glucose oxidase retained the biocatalytic activities and glucose is oxidized by dissolved oxygen efficiently, which is catalyzed by glucose oxidase to produce hydrogen peroxide. The exhibited good linear behavior in glucose concentrations ranging from 3.30 x 10(-6) M to 5.56 x 10(-2) M with a low detection limit of 3.61 x 10(-7) M. Moreover, the fabricated biosensor exhibited long-term stability, good reproducibility, and absence of interference from other co-existing electroactive species. Thus, the facile and effective methodology of sensor preparation in this study will promote further electrochemical research on proteins, biosensors, and other bioelectrochemical devices. (c) 2013 Elsevier B.V. All rights reserved.