In this study, we present the use of a Ni-Cu alloy electrodeposited on a titanium electrode as a sensor for non-enzymatic glucose oxidation. Electrochemical parameters such as bath composition, potential, and deposition times used to obtain the films were studied. After optimization of operating conditions, the morphology, chemical composition, and surface structure of the Ni-Cu alloy electrodes were characterized using scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX) and X-ray diffraction spectroscopy (XRD). Electrocatalytic activity of the modified Ni-Cu electrodes towards non-enzymatic glucose oxidation was then evaluated by cyclic voltammetry and chronoamperometric and impedance spectroscopy. The experimental impedance data were fitted with an equivalent circuit model representing the electrode/electrolyte interface, to evaluate the charge transfer rate as well as the electrical characteristics of the catalytic surface. Cyclic voltammetry experiments show that the Ni-Cu films prepared at -0.975 V for 10 min significantly generated the highest oxidation current, extended linear responses for glucose concentrations in the range of 0.25 to 8 mM, with excellent values of the limit of detection (LOD, 4.97 µM) and sensitivity ( 546 µA mM -1 cm -2 ). Effects of endogenous interfering species (ascorbic acid (AA), uric acid (UA), oxalic acid, fructose, sucrose, NaCl, and KNO 3 ) on the electrocatalytic response of Ni-Cu/Ti were also studied and showed that Ni-Cu/Ti is a selective electrocatalyst towards glucose oxidation. The application of Ni-Cu/Ti biosensor in real human saliva and urine samples is demonstrated using chronoamperometric detection: the increase in current remains proportional to successive additions of glucose. Finally, it was shown that the Cu-Ni/Ti sensor presented the lowest charge transfer resistance (R ct ) and the highest double layer capacitance values compared to those of the pure Ti, Ni/Ti, and Cu/Ti electrodes. The improved electrocatalytic activity is mainly attributed by the synergistic effect between Ni and Cu.