Abstract The electrodeposition method was firstly applied to obtain uniform cube‐shaped copper nanoparticles on conductive glass (ITO), and then a layer of tiny nickel nanoparticles. A bimetallic composite electrode (Cu−Ni/ITO), characterized by TEM, XPS and XRD, was prepared to construct the non‐enzyme electrochemical glucose sensor with high catalytic activity. The catalytic performance of Cu−Ni/ITO had been greatly improved, probably due to the synergistic bimetallic catalysis effect. The electrode had a satisfactory linear response in the range of 2.5×10 −7 M to 2.6×10 −3 M, with detection limit as low as 67 nM. Besides, Cu−Ni/ITO had good anti‐interference ability and reproducibility, indicating the promising application for glucose detection in practical samples.