A highly sensitive and stable electrochemical non-enzymatic sensor was developed by modified glassy carbon electrode with Au-CuO nanocomposites. The composite material was synthesized through co-precipitation method and characterized by field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDAX) and X-ray diffraction (XRD). The prepared composite was directly used as electrochemical sensor of glucose and its electrocatalytic activity was evaluated by cyclicvoltammetry (CV) and chronoamperometry (CA). In 0.1 M NaOH alkaline medium the modified (Nf/Au-CuO/GCE) electrode shows high electrocatalytic activity towards the oxidation of glucose. The amperometric result was recorded at an applied potential of +0.6 V for Nf/Au-CuO/GCE with a high sensitivity (3126.76 μAmM−1cm−2), wide linear range (5 μM–650 μM), fast current response time (3 s), and excellent low detection limit (1.4 μM, S/N = 3). In addition, the proposed electrode also proved excellent selectivity related to glucose in the presence of common interfering species such as ascorbic acid (AA), dopamine (DA) and uric acid (UA) as well as good long-term stability which makes it to determine the glucose in real human urine samples. © 2018 Elsevier Ltd