A fluorescence-based immunochromatographic assay (ICA) for fumonisin B1 (FB1) that employs conjugates of fluorescent microspheres and monoclonal antibodies (FM-mAbs) as detection reporters is described. The ICA is based on the competitive reaction between FB1-bovine serum albumin (BSA; test line) and the target FB1 for binding to the FM-mAb conjugates. A limit of detection (LOD) for FB1 of 0.12 ng/mL was obtained, with an analytical working range of 0.25-2.0 ng/mL (corresponding to 250-2000 μg/kg in maize flour samples, according to the extraction procedure). The recoveries of the ICA to detect FB1 in maize samples ranged from 91.4 to 118.2%. A quantitative comparison of the fluorescence-based ICA and HPLC-MS/MS analysis of naturally contaminated maize samples indicated good agreement between the two methods (r(2) = 0.93). By replacing the target of interest, the FM-based ICA can easily be extended to other chemical contaminants and thus represents a versatile strategy for food safety analysis.