A compact and intimate interfacial contact between the modified film and the conducting electrode is crucial for electrochemical biosensors. The direct drop-casting of nanomaterials onto the working electrode often fails to construct a compact interfacial arrangement, which results in sluggish electrode kinetics. Here, we describe a simple and cost-effective strategy to produce CuO nanostructure using a modified hydrothermal route. The in-situ growth allowed the formation of a highly ordered interconnected network of sharp flakes configured in the form of large spheres with excellent ITO surface coverage. The CuO nanostructures were highly electrochemically active toward the oxidation of β–adrenergic agonists, i.e., formoterol fumarate (FF). The analytical ability was studied by comparison of the electrochemical behavior of ITO based electrode with its glassy carbon electrode counterpart. The binder-less CuO-based ITO electrode successfully determined FF with a detection window of 0.01 µM to 0.46 µM with practical application for real broiler feed samples collected from the local poultry farms in Hyderabad, Pakistan.