This research aims at elaborating on the construction of a novel nanostructured copper oxide (Nano-CuO) sputtered thin film on the conductive fluorinated-tin oxide (FTO) layer that was exploited to immobilize glucose oxidase (GOx) enzyme via chitosan for developing impedimetric glucose biosensing. The distinct Nano-CuO thin film was fabricated by reactive DC magnetron sputtering system at the optimized instrumental deposition conditions. The FTO/Nano-CuO/Chitosan/GOx biosensor containing several layers afforded excellent microenvironment for rapid biocatalytic reaction to glucose. Field emission-scanning electron microscopy (FE-SEM), Ultraviolet-Visible spectroscopy (UV-Vis), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) were established the morphology of modified electrodes surface and electrochemical behavior of glucose on the fabricated biosensor. Characterization of the surface morphology and roughness of CuO thin film by FE-SEM exhibits cavities of the nanoporous film as an effective biosensing area for covalent enzyme immobilization. Invented FTO/Nano-CuO/Chitosan/GOx biosensor was employed for glucose determination using the impedimetric technique. The impedimetric outcomes display high sensitivity for glucose (0.261 k Omega per mM) detection within 0.2-15 mM and limit of detection as 27 mu M. The declared biosensor has been applied as a careful, noncomplicated, and exact biosensor for recognition of glucose in the real samples analysis. (C) 2018 Elsevier B.V. All rights reserved.