Copper nanoclusters (Cu NCs) have emerged as promising nano-emitters. However, their limited luminescence stability and emission intensity restrict their application in electrochemiluminescence (ECL). Herein, a ligand regulation strategy was developed to enhance the anodic ECL of Cu NCs. Specifically, we utilized ovalbumin (OVA), a naturally occurring protein, as a stabilizing ligand for the Cu NCs and introduced terbium ions into the ligand. This approach optimizes ECL performance by enhancing electron transfer efficiency and suppressing non-radiative relaxation. Additionally, we used the enhanced ECL nano-emitter as a signal probe and combined it with split aptamers to develop an analytical platform. This platform is designed for the detection of enrofloxacin (ENR) in food samples. The sensing platform incorporated a self-designed ENR test cell combined with 3D printing, ITO sputtering and screen-printing technologies, which greatly improved the reproducibility of the test results. Our study highlights the potential of this innovative detection method in the realm of food detection.