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Metal–organic frameworks (MOFs) have broad potential applications in electrochemical glucose detection. Herein, a green ultrasonic synthesis process is presented for preparing two-dimensional (2D) copper–nickel metal–organic framework nanosheets (CuNi-MOFNs) for glucose detection. The synthesized CuNi-MOFNs were characterized using scanning electron microscopy (SEM), scanning transmission electron microscope (STEM), X-ray diffractometer (XRD), and X-ray photoelectron spectrometer (XPS). The CuNi-MOFN nanocomposites were used to cover the glassy carbon electrode (GCE) and the CuNi-MOFNs-modified electrode was studied in alkaline media. Cyclic voltammetry (CV) and amperometric i–t curves indicated that the CuNi-MOFNs-modified electrode revealed great electrochemical performances towards glucose oxidation. Due to the ease of access to active metal sites in large specific surface of nanosheets, the CuNi-MOFNs-modified electrode can effectively improve the electronic transfer rate and enhance electrocatalytic activity of the CuNi-MOFNs-modified electrode. The CuNi-MOFNs-modified electrode showed electrochemical performances for glucose detection with a linear range from 0.01 mM to 4 mM, sensitivity of 702 μAmM⁻¹cm⁻², and detection limit of 3.33 μΜ (S/N = 3). The CuNi-MOFNs-modified electrode exhibited excellent anti-interference ability and high selectivity in glucose measurements. Hence, the CuNi-MOFNs-modified electrode has good, promising prospects in non-enzymatic electrochemical glucose detection.