Diabetes is a major worldwide health issue, impacting millions of people around the globe and putting pressure on healthcare systems. Accurate detection of glucose is critical for efficient diabetes care, because it allows for prompt action to control blood sugar levels and avoid problems. Reliable glucose-sensing devices provide individuals with real-time information, allowing them to make more educated food, medicine, and lifestyle decisions. The progress of glucose sensing holds the key to increasing the quality of life for diabetics and lowering the burden of this prevalent condition. The present investigation addresses the synthesis of a CuO@lemon-extract nanoporous material using the sol–gel process. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to analyze the morphological properties of the composite, which revealed a homogeneous integration of CuO nanoparticles (NPs) on the surface of the matrix. The existence of primarily oxidized copper species, especially CuO, was confirmed by X-ray diffraction spectroscopy (XRD) investigation in combination with energy-dispersive X-ray (EDX) spectroscopy. The CuO@lemon-extract-modified glassy carbon electrode (CuO@lemon-extract GCE) performed well in non-enzymatic electrochemical sensing applications such as differential pulse voltammetry (DPV) and amperometric glucose detection. The electrode achieved a notable sensitivity of 3293 µA mM−1 cm−2 after careful adjustment, with a noticeable detection limit of 0.01 µM (signal-to-noise ratio of 3). The operational range of the electrode was 0.01 µM to 0.2 µM, with potential applied of 0.53 V vs. Ag/AgCl. These findings underscore the CuO@lemon-extract GCE’s promise as a robust and reliable platform for electrochemical glucose sensing, promising advances in non-enzymatic glucose sensing (NEGS) techniques.