The present work demonstrates a novel, simple approach for fabrication of sensitive and selective glucose biosensors based on polypyrrole (PPy) nanofibers without using price-rising mediator or nanoparticles. The nanofibers were produced by chemical vapor polymerization of pyrrole on FeCl3 containing PAN nanofiber mats. Amperometric biosensors were constructed by entrapment of glucose oxidase on the nanofibers with the help of glutaraldehyde. The operational parameters such as pH, working potential, enzyme amount were optimized. For every biosensor sensitivity, linear range, limit of detection (LOD), and Michaelis-Menten constant values were determined and the stabilities of some of the sensors were tested. Our studies underlined the clear effect of enzyme content where the biosensors tend to show widened the linear detection range as the enzyme loadings increased +0.65V. PPy-NFs/GOx-1 biosensor showed good sensitivity (68.95 mu A/mM.cm(2)) and LOD (7.8 mu M) without any interference effect at +0.65V. On the other hand, when it was operated at -0.65V (oxygen consumption) the biosensor exhibited enhanced sensitivity of 116.42 mu A/mM.cm(2) with modest LOD and operational stability. Due to utilization of biocompatible, large surface area, nanoporous PPy nanofibers; an improved analytical performance was achieved with a very short response time, good stability and high selectivity, which make them feasible candidates for commercialization. (C) 2017 The Electrochemical Society. All rights reserved.