Technical Abstract: Deoxynivalenol (DON) is a toxin produced by certain species of fungi that can infest wheat, barley, and corn. The fungi cause diseases in crops worldwide and some of the secondary metabolites, such as DON, can adversely affect animal health and food safety. To rapidly monitor DON in wheat a biosensor using the principle of biolayer interferometry (BLI) was developed. Signal from the sensor was substantially amplified through the use of a primary antibody-colloidal gold conjugate. The amplification was much greater in the presence of wheat matrix than in buffered solution, suggesting matrix components may have contributed to the enhancement. The improved signal provided by the amplification allowed for the development of rapid qualitative and quantitative assays. The limit of detection of the method was 0.09 mg/kg and the limit of quantitation was 0.35 mg/kg. Recovery from wheat spiked over the range from 0.2 to 5 mg/kg averaged 103% (RSD 12%). The quantitative assay compared favorably (r2 = 0.9698) to a reference chromatographic method for 40 naturally contaminated wheats. The qualitative assay was able to accurately classify the same group of samples as either above or below a 0.5 mg/kg threshold. These results suggest that the BLI technique can be used to rapidly measure DON in wheat.