Highly sensitive low-cost non-enzymatic glucose sensor of copper oxide (CuO) nanorods (NRs) was successfully fabricated using a two-step anodization method with different voltages to generate Cu(OH)2 nanowires. CuO NRs were created by annealing Cu(OH)2 samples at 200 °C for 3 h. The nanostructures were analyzed by X-ray diffraction (XRD), field-emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX), and Fluorescence Luminescence (FL). Based on the XRD pattern, the CuO NRs showed a monoclinic structure with an average crystallite size (17. 8–49.6 nm). Also, FE-SEM presented nanorod-like shapes with a particle size range of (10–30 nm), (30–35 nm), and (40–60 nm) for voltage anodization (4, 5, and 6), respectively with thickness between 653.2 nm and 1.530 μm. FL emission spectra were observed in the range of 380–383 nm. This referred to an expansion in the energy gap (3.22–3. 24 eV). The structure showed a high activity of glucose detection with a sensitivity of (502.29, 650.4, and 783.3 μA Mm−1 cm−2) with LOD (0.45, 0.35, and 0.5 μA), respectively. Furthermore, the CuO electrode demonstrated high thermal stability, suggesting the applicability of the device for bio-electrochemical applications in the future.