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The analysis of chemical xenobiotics in human, food, and environmental samples has become a global priority. Consequently, both public and private laboratories require rapid, cost-effective analytical methods for quality and safety control. Fluopicolide, a fungicide used to combat plant diseases, poses potential toxicological risks to humans and animals. In this study, the first monoclonal antibodies targeting this fungicide were generated, and a fast, highly sensitive immunoanalytical method was developed. With this aim, two haptens with opposite linker tethering sites were rationally designed and synthesized in high yields for immunogen and antigen preparation. Both haptens closely mimic fluopicolide, and their conjugates elicited similar immunogenicity in rabbits. Furthermore, a collection of high-affinity monoclonal antibodies was obtained from mice immunized with either of the two immunizing conjugates. A third hapten, featuring a heterologous linker tethering site and an additional chlorinated phenyl position, was also synthesized. All antibodies were thoroughly characterized using two competitive enzyme-linked immunosorbent assay formats, utilizing both homologous and heterologous conjugates. Remarkably, outstanding sensitivity to fluopicolide was achieved (IC 50  = 0.02 ng/mL) when the hapten conjugate with double heterology was used as the competing antigen. This immunoassay was optimized and successfully applied to the analysis of fortified tomato and grape samples across a concentration range of 1-2000 ng/mL, yielding excellent recoveries (77.7 %-119.6 %) and coefficients of variation below 20 %. These findings demonstrate the effectiveness of the developed immunoassay for the analysis of fluopicolide residues in food samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)