Cyprodinil is a new-generation anilinopyrimidine fungicide widely used in crop protection and frequently found in fruits. In this study, novel derivatives of cyprodinil with linker site heterologies were synthesized and employed in order to produce antibodies with enhanced affinity. Moreover, moiety-heterologous haptens were designed and prepared for assay sensitivity improvement. Two competitive enzyme-linked immunosorbent assays for the analysis of this active substance were developed using direct and indirect formats, achieving IC50 values around 0.15 μg/L. Analytical figures of merit and usability of the optimized assays were evaluated with wine and cider as model food processed matrices. The obtained recoveries were from 90% to 120%, and the limit of quantification was in the 1–5 μg/L range. Finally, a monitoring study (n = 150) was performed to estimate the occurrence and the concentration of cyprodinil in commercial wine and cider products from different origins. We found that 28% of the analysed wine samples contained cyprodinil residues at levels higher than 5 μg/L. •Regioisomeric haptens with alternative linker sites were prepared for immunization.•Moiety-heterologous haptens were synthesized for immunoassay sensitivity enhancement.•High-sensitivity assays for cyprodinil were developed, with IC50 values below 1 μg/L.•Direct and indirect cELISAs were optimized for trace analysis in wines and cider.•Residues were encountered in 28% of commercial wine samples from different origins.