In the present work, a template-free single-step fabrication of Lithium perchlorate (LiClO4) doped 1-D Polypyrrole nanofibers network (PNN) (without using electrospinning) toward glucose biosensing application has been reported. The PNN with different concentrations of LiClO4 was grown over the Platinum coated glass substrate by electropolymerization method and utilized as a support matrix for immobilization of enzyme. The effect of LiClO4 concentration on glucose biosensor performance has been demonstrated for the first time. The modification in morphology and reduction in charge transfer resistance of PNNs by varying LiClO4 concentration were found to play a significant role in the improved figure of merits of the biosensor. Among all the types of as-fabricated PNN electrodes (prepared by using different concentrations of LiClO4 viz. 1, 10, 50, and 70 mM) the best response was obtained corresponding to highest LiClO4 concentration. The enzyme loading and charge transfer resistance were drastically improved by approximately three folds owing to the higher surface area and improved conductivity of PNN electrodes thereby resulting in ten-fold increment in sensitivity. The as-prepared biosensor showed the highest sensitivity of 4.34 mA-cm(-2)-M-1 and a linear range of 0.1-4.6 mM with good stability and high selectivity for glucose detection. (C) 2018 The Electrochemical Society.