© 2019 Elsevier B.V.In this work, graphite pencil electrode (GPE) was modified with Cu nanoparticles (CuNPs) for enzyme-free flow injection (FI) amperometric detection of glucose. To modify electrode, CuNPs were electrodeposited onto GPE surface by recording 10 successive cyclic voltammograms (CVs) of 2.0 mM Cu(NO3)2 in 100 mM KNO3. The electrochemical characterizations of the electrodes were realized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry techniques. The SEM images and EDX spectra of the pencil leads were recorded to investigate the surface morphologies of the electrodes. Then, the electrochemical response of both unmodified GPE and CuNPs/GPE towards electrochemical oxidation of glucose was performed by recording CVs in the supporting electrolyte consisted of 100 mM NaOH and 100 mM KCl. CVs recorded at GPE showed that the electron transfer rate of glucose is relatively slow and oxidation of glucose irreversibly observed with very low current at a high positive potential (+0.70 V). On the other hand, glucose oxidized at more negative potential value (+0.45 V) than that of unmodified GPE with a view of sharp peak at CuNPs/GPE. These results show that CuNPs/GPE exhibits excellent electrocatalytic activity and high electrochemical response towards oxidation of glucose because electron transfer rate of glucose was remarkably enhanced by modification of GPE with CuNPs. In the flow injection (FI) amperometric experiments, +0.45 V and 2.5 mL/min were used as optimal values for applied potential (Eap) and flow rate (fr), respectively. A linear calibration curve was obtained in the range from 0.10 to 400 µM glucose with a detection limit and sensitivity of 0.04 µM and 0.830µA µM−1 cm−2, respectively. The selectivity of glucose sensor was tested in the presence of various interferences. At last step, constructed glucose biosensor was successfully tested on some real glucose samples.