This article describes the synthesis of nanostructured copper oxide on copper wires and its application for the detection of hydrogen peroxide. Copper oxide petal nanostructures were obtained by a one-step hydrothermal oxidation method. The resulting coating is uniform and dense and shows good adhesion to the wire surface. Structure, surface, and composition of the obtained samples were studied using field-emission scanning electron microscopy along with energy-dispersive spectroscopy and X-ray diffractometry. The resulting nanostructured samples were used for electrochemical determination of the H2O2 content in a 0.1 M NaOH buffer solution using cyclic voltammetry, differential pulse voltammetry, and i–t measurements. A good linear relationship between the peak current and the concentration of H2O2 in the range from 10 to 1800 μM was obtained. The sensitivity of the obtained CuO electrode is 439.19 μA·mM−1. The calculated limit of detection is 1.34 μM, assuming a signal-to-noise ratio of 3. The investigation of the system for sensitivity to interference showed that the most common interfering substances, that is, ascorbic acid, uric acid, dopamine, NaCl, glucose, and acetaminophen, do not affect the electrochemical response. The real milk sample test showed a high recovery rate (more than 95%). According to the obtained results, this sensor is suitable for practical use for the qualitative detection of H2O2 in real samples, as well as for the quantitative determination of its concentration.