As an important signal molecule, nitric oxide (NO) is involved in a variety of physiological and pathophysiological processes, which makes real-time NO detection imperative in the physiological environment. However, probing NO in physiological systems remains a great challenge due to its short half-life, low concentration, rapid diffusion, and easy interference by other chemicals. To be noted, traditional detection methods often require time-consuming sample pretreatment and cannot be used for continuous monitoring in vivo. Herein, we report copper oxide-decorated reduced graphene oxide (rGO/CuO) synthesized via a facile and eco-friendly solvothermal method. By taking advantage of the synergetic effect of CuO and rGO, the rGO/CuO sensor demonstrates impressive electrochemical performance for NO detection with a low detection limit (9.57 nM), wide linear range (90 nM to 138 μM), and high sensitivity (5.48 μA μM⁻¹ cm⁻²). More importantly, the prepared sensor also shows excellent selectivity, repeatability, reproducibility, long-term operational stability, and fast response for NO detection, which meets the requirements of monitoring of NO released from living cells. Therefore, we believe that the reported rGO/CuO sensor has great potential for in vivo detection and even clinical diagnosis.