Non-enzymatic glucose sensors based on metal oxides are receiving remarkable attention owing to their outstanding characteristics of being easy-to use, low cost, and reusability. However, the disadvantage of weak anti-interference associated with poor selectivity significantly restricts their applicability. Herein, we report a two-step in situ fabrication of nanosized CuO encapsulated Ni/Co bimetal Prussian blue (PB) with a typical core–shell structure, which can be efficiently used for non-enzymatic glucose detection, ascribing to the permeability and abundant active sites of out-shelled crystalline porous Ni/Co PB and the high catalytic activity and conductivity of embedded CuO nanoparticles, afforded by their mutual synergistic interactions. The glassy carbon electrode modified with the hybrid of the CuO-encapsulated Ni/Co PB (simplified as the Ni/Co-PB/CuO/GCE electrode) exhibited a high glucose sensitivity of 600 μA mM⁻¹ cm⁻² with a low detection limit of 0.69 μM (S/N = 3), a fast response time (less than 3 s), and excellent long-term stability. In addition, the CuO-encapsulated Ni/Co PB showed favorable anti-interference ability in the presence of ascorbic acid (AA), L-lysine (Lys), dopamine (DA), cysteine (Cys), dopamine (DA), and KCl interferences. The reusability and long-term stability, as well as the practicability of the Ni/Co-PB/CuO/GCE sensing electrode verified by testing real serum samples were also investigated, and the experimental results demonstrated the applicability of the core–shell NiCo-PB/CuO based flexible electrochemical sensor for non-enzymatic glucose sensing in practical applications.