Immunochemical methods have been consolidated during the last few years as complementary analytical strategies for chemical contaminant and residue determination. However, generation of suitable immunoreagents for small organic molecules demands adequate hapten design. In this study, fenhexamid was considered as a model compound and novel haptens were designed and synthesized in order to evaluate the influence of the linker tethering site on antibody binding properties and immunoassay parameters. Haptens were conceived with the spacer arm at different positions, while the more antigenic aromatic moiety was kept free. The synthesis of these functionalized compounds was accomplished by total construction of the molecule through several steps. This strategy afforded very high-affinity monoclonal antibodies specific of fenhexamid, with IC50 values around or below 0.1 nM. Using these novel immunoreagents, a direct competitive enzyme-linked immunosorbent assay with a remarkably low limit of detection (4 ng L-1) was developed for the determination of fenhexamid residues. The selected immunoassay was investigated in terms of trueness, precision, repeatability, and robustness. The QuEChERS extraction methodology was applied to fortified samples and recoveries between 83% and 113%, with relative standard deviations below 20%, were observed. Moreover, contaminated and blind spiked samples were measured by the developed immunoassay and by ultra-performance liquid chromatography coupled to tandem mass spectrometry, showing statistically comparable results.