Abstract Highly ordered TiO2 nanotube arrays (TiO2NTs) evenly modified by Ni–Cu nanoparticles were successfully prepared by potential step method. Their morphologies, structures, and alloy composition were characterized by FESEM, XRD and EDS, respectively. The as-prepared Ni–Cu/TiO2NTs electrodes were employed for non-enzymatic glucose detection in alkaline electrolyte and showed better electro-catalytic activity compared with Ni/TiO2NTs and Cu/TiO2NTs electrodes. Factors that affected the electrocatalysis of the electrodes were examined and optimized. Consequently, a sensitive amperometric electrode of glucose was achieved under 0.6V vs. Ag/AgCl with a high sensitivity (1590.9μAmM−1cm−2), low detection limit (5μM) and wide linear range from 10μM to 3.2mM (R2=0.993). Furthermore, the oxidable species such as ascorbic acid and uric acid showed no significant interference in determination of glucose. The experiment results revealed a very good reproducibility and high stability for the proposed Ni–Cu/TiO2NTs electrodes.