Tetracycline antibiotics (TCs) are widely used in medicine, agriculture, and animal husbandry. However, their overuse has led to environmental pollution, posing a significant threat to water sources, soil, and food safety. Therefore, there is an urgent need for efficient, sensitive, simple, and low-cost detection methods for environmental pollution monitoring. In this study, the catalytic activity of copper-based nanozymes was regulated by AAs. Lysine, aspartic acid, glycine, and arginine were chosen as ligands to synthesize different copper-based nanozymes. The results showed that the type of amino acid significantly influenced the particle size, morphology, and peroxidase (POD)-like catalytic activity of Cu2O. Based on these amino acid-regulated Cu2O nanozymes, we further developed a highly sensitive, easy-to-use, and low-cost colorimetric sensor array that can effectively distinguish TCs. This sensor array was successfully validated in binary mixtures and wastewater environments. This study not only provides important insights into the small-molecule regulation of copper-based nanozyme catalytic performance but also offers a novel approach for the detection of TCs in environmental monitoring.