<p class="PaperAbstract">Cu<sub>x</sub>O-NiO nanocomposite film for the non-enzymatic determination of glucose was prepared by the novel modifying method. At first, anodized Cu electrode was kept in a mixture solution of CuSO<sub>4</sub>, NiSO<sub>4</sub> and H<sub>2</sub>SO<sub>4</sub> for 15 minutes. Then, a cathodization process with a step potential of -6 V in a mixture solution of CuSO<sub>4</sub> and NiSO<sub>4</sub> was initiated, generating formation of porous Cu-Ni film on the bare Cu electrode by electrodeposition assisted by the release of hydrogen bubbles acting as soft templates. Optimized conditions were determined by the experimental design software for electrodeposition process.<strong> </strong>Afterward, Cu-Ni modified electrode was scanned by cyclic voltammetry (CV) method in NaOH solution to convert Cu and Ni nanoparticles to the nano-scaled Cu<sub>x</sub>O-NiO film. The electrocatalytic behavior of the novel Cu<sub>x</sub>O-NiO film toward glucose oxidation was studied by CV and chronoamperometry (CHA) techniques. The calibration curve of glucose was found linear in a wide range of 0.04–5.76 mM, with a low limit of detection (LOD) of 7.3 µM (S/N = 3) and high sensitivity (1.38 mA mM<sup>-1</sup> cm<sup>-2</sup>). The sensor showed high selectivity against some usual interfering species and high stability (loss of only 6.3 % of its performance over one month). The prepared Cu<sub>x</sub>O-NiO nanofilm based sensor was successfully applied for monitoring glucose in human blood serum and urine samples.</p>