Enrofloxacin, a fluoroquinolone widely used in animal husbandry, poses significant risks to human health and the environment due to residue accumulation and antibiotic resistance. Aptamer-based electrochemical sensors provide the real-time measurement of the biomolecules from the samples, making them a powerful technique in clinical and research applications in recent years. This study presents the development of a novel, label-free electrochemical aptasensor for the sensitive and selective detection of enrofloxacin. The sensor utilizes a reduced graphene oxide–gold nanoparticle (rGO–AuNP) composite, which provides a high surface area, enhanced electron transfer, and excellent conductivity for aptamer immobilization. The rGO-AuNP nanocomposite was characterized using UV-Vis spectroscopy, scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy, while its electrochemical properties were evaluated using cyclic voltammetry and differential pulse voltammetry. The recovery rate of enrofloxacin residues in milk samples was 92.11–115.51%. Demonstrating its potential applications in food safety control. The aptasensor exhibited high sensitivity, selectivity, and repeatability for enrofloxacin detection, with minimal interference from other antibiotics. Its performance highlights its potential as a cost-effective and efficient tool for food safety applications.