Fumonisin B1, a mycotoxin produced by certainFusariummoulds, consists of two tricarballylic acid groups esterified to a 20‐carbon backbone. Under alkaline conditions, or through metabolism, the aminopentol backbone, also known as hydrolysed Fumonisin B1(HFB1) can be formed and is itself cytotoxic. Although the occurrence of HFB1in corn‐based foods is suspected, because of the ubiquitous nature of FB1in corn, analytical methods for its detection are difficult. In the present report we describe a monoclonal antibody‐based competitive direct enzyme‐linked immunosorbent assay (CD‐ELISA) for the rapid analysis of HFB1in corn. The concentration required to inhibit enzyme conjugate binding by 50% (IC50) was 36 ng/ml. The limit of detection of HFB, by the CD‐ELISA was 2 ng/ml, when HFB1was added in bovine serum albumin—phosphate buffered saline. The antibody also cross‐reacted with the hydrolysis products of FB2, FB3, and FB4, having IC50values of 331, 174, and 1700 ng/ml respectively. The antibody did not react with the intact fumonisins, sphingosine, sphinganine, or tricarballylic acid. Samples of corn spiked with HFB1over the range of 5–1000 ng/g indicated the CD‐ELISA has a limit of detection of 5 ng/g and an IC50of 41 ng/g in this matrix. The CD‐ELISA provides a sensitive and rapid tool for the analysis of corn‐based foods for HFB1.