With diabetes mellitus increasing, developing non-enzymatic glucose sensors to replace enzyme-based sensors has become more urgent, owing to its intrinsic disadvantages of enzymes. Herein, CuO microflowers were successfully synthesized through a green hydrothermal method with no template or surfactant. X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy and transmission electron microscopy were used to study the crystalline structure, elemental composition and morphology of the as-obtained sample. Then, the CuO microflowers acted as non-enzymatic glucose sensor electrode material. The electrochemical test results indicate that the glucose oxidation controlled by diffusion at 0.5 V had a short response time(5 s), the linear range was wide (∼11 mM), and the detection limit was low(1.3 µm). Moreover, the CuO microflowers exhibit great selectivity to glucose and have high poison resistance to chloride ions. Thus, all these results imply that the as-fabricated CuO has a great application prospect for nonenzymatic glucose sensor electrode material.