© 2020 Elsevier B.V.This study reports non-ezymatic electrocatalytic amperometric glucose biosensor based on a graphite rod electrode (GRE) modified with biomimetic-composite consisting of Ni nanoparticles (Ni-NPs) and polypyrrole (Ppy) prepared by 1 cycle electro polymerization of pyrrole monomer (Ni-NPs/Ppy(1)/GRE). During the modification of GRE, the electropolymerization of pyrrole and the electrodeposition of Ni-NPs onto GRE surface were consequentially performed by potential cycling. Surface morphology of Ni-NPs/Ppy(1)/GRE electrode was evaluated by atomic force microscopy and scanning electron microscopy based imaging, and electrochemical characterization of electrodes was performed by electrochemical impedance spectroscopy and cyclic voltammetry. Cyclic voltammograms recorded in the presence of glucose show that Ni-NPs/Ppy(1)/GRE at + 500 mV vs Ag/AgCl exhibits efficient electrocatalytic oxidation activity towards glucose, while the oxidation of glucose was not observed at a bare GRE. Amperometric sensing of glucose was performed by Ni-NPs/Ppy(1)/GRE at constant + 450 mV vs Ag/AgCl electrode potential in 0.10 mol L−1 NaOH. Ni-NPs/Ppy(1)/GRE-based sensor, which was characterized by a wide linear glucose determination range between 1.0 and 1000 µmol L−1 with a limit of detection of 0.4 µmol L−1 and a sensitivity of 2873 µA mmol−1 L cm−2. The applicability of here reported Ni-NPs/Ppy(1)/GRE-based sensor has been demonstrated by the determination of glucose concentrations in real samples.