Abstract: Many studies about providing suitable coating on the electrodes have been investigated. Electron transfer chemical and electrocatalysis properties are very important for them. Exact determination of glucose concentration is necessary for treatment of diabet and food industry.A novel Ni (Nickel)/CS(chitosan)/CNT(carbon nanotube) network nanocomposite was constructed on graphite electrode for glucose detection. Firstly, multi-walled carbon nanotubes were purified and characterized by Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM).Chitosan include excellent biocompatibility, biodegradability, nontoxicity, high mechanical strength, good adhesion and cheapness properties, therefore it has been used as an immobilization matrix. Although it has poor electrical conductivity, but it usually has been combined with carbon nanotubes, redox mediator and metal nanoparticles. Carbon nanotubes have been used in various sensors due to their unique properties such as high electronical and mechanical stability. Nickel hydroxide severely adsorbs some organic substances such as chitosan and has high electrocatalytical properties. Therefore, it has been used for preparation of modified electrodes. In this work, a film of Ni/CS/CNTs network was prepared for oxidation of glucose. The modified electrode with the Ni/CS/CNTs showed excellent electrocatalytical response to the oxidation, which was studied by cyclic voltammetry (CV). The synthesized biosensor exhibited rapid response, low detection limit, broad linear range, good reproducibility and stability.