A multilayered glucose biosensor with enhanced electron transport was fabricated via the sequential electrodeposition of chitosan gold nanocomposite (CGNC) and nickel hydroxide (Ni(OH)(2)) on a bare gold electrode and subsequent immobilization of glucose oxidase. A thin film of Ni(OH)(2) deposited on CGNC modified gold electrode serves as an electrochemical redox probe as well as a matrix for the immobilization of glucose oxidase retaining its activity. Electron transport property of CGNC has been exploited to enhance the electron transport between the analyte and electrode. Electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Under optimal conditions the biosensor exhibits a linear range from 1 mu M to 100 mu M with a limit of detection (lod) down to 100 nM. The sensor shows a low Michaelis-Menten constant value of 2.4 mu M indicates the high affinity of enzyme to the analyte points to the retained activity of enzyme after immobilization. The present glucose sensor with the high selectivity, sensitivity and stability is promising for practical clinical applications. (C) 2013 Elsevier Ltd. All rights reserved.