This study presents an innovative approach for the detection of 5-nitroimidazole antibiotics, namely ornidazole, secnidazole, metronidazole and tinidazole. These antibiotics are widely used to treat microbial infections in both humans and livestock industries. Their unregulated use poses environmental and health risks due to their persistence in water bodies and potential to contribute to drug resistance. In response, this research proposes the utilisation of the self-aggregation property of a single unmodified non-aromatic amino acid, ʟ-lysine, for the detection of these antibiotics. Lysine exhibits strong fluorescence in water in a concentration-dependent manner and the interaction of lysine with 5-nitroimidazole antibiotics resulted in the quenching of its fluorescence. With this technique, we obtained a detection limit of 2.5 μM for metronidazole (MDZ), 2.65 μM for ornidazole (ODZ), 3.13 μM for secnidazole (SDZ) and 2.98 μM for tinidazole (TDZ). This cost-effective approach also offers practicality through visual detection on paper strips as well as successful application in detecting those antibiotics in milk samples and animal products. Taking advantage of the unique properties of lysine and its interaction with antibiotics, this method provides a robust, simplified yet efficient means of detecting 5-nitroimidazole antibiotics, which holds significant potential for real-world implementation. © 2023 Elsevier B.V.