Mimotopes, or epitope mimics, can be applied to competitive immunoassays, for the detection of low molecular weight natural toxicants, as an alternative to toxin-conjugates. In this work, we report the development of a microarray-based immunoassay for the detection of fumonisin B1 using a novel mimotope selected by phage display technology. Fumonisin-specific antibody was used to isolate mimotopes from a 12-mer peptide library in successive selection rounds. Enrichment of antibody binding phages was observed after three panning rounds, and sequence analysis of randomly selected monoclonal phages revealed two conserved peptide sequences. Clone A2, with peptide sequence VTPNDDTFDPFR, showed the best response in enzyme-linked immunosorbent assay (ELISA) in terms of sensitivity and reproducibility and was selected for microarray development. A biotinylated synthetic derivative of this mimotope was immobilized onto epoxy-glass slides, and fumonisin B1 was detected in a competitive binding inhibition assay using the antifumonisin antibody and a labeled secondary antibody. The array showed an IC50 value of 37.1 ± 2.4 ng mL-1 (n = 9), a detection limit of 11.1 ng mL-1, and a dynamic range from 17.3 to 79.6 ng mL-1. Good specificity toward fumonisin B1 and its structural analog, fumonisin B2, was observed, together with negligible cross-reactivity for other mycotoxins produced by the same fungi species. The mimotope microarray was applied to the analysis of fumonisin B1 in spiked maize and wheat samples. The method enabled quantification of the mycotoxin at the levels set by European legislation and holds promise for future adaptation to include other mycotoxins for multiplex detection.