Technical Abstract: Biolayer interferometry allows for the real time monitoring of the interactions between molecules without the need for reagents with enzymatic, fluorescent, or radioactive labels. The technology is based upon the changes in interference pattern of light reflected from the surface of an optical fiber that can occur when materials bind to the tip of the fiber. In this report a deoxynivalenol-bovine serum albumin (DON-BSA) conjugate was non-covalently attached to the surface of aminopropylsilane sensors and the change in interference pattern resulting from the binding of DON-specific antibodies was measured. The assay format was competitive, with larger concentrations of DON causing greater inhibition of binding of antibodies to the sensor surface. The technique was used to measure DON in spiked whole wheat flour, with a limit of detection of 0.10 mg DON/kg. Matrix interferences were an issue, and adequate quantification required using matrix-matched standards. Recoveries at five spiking levels over the range from 0.2 to 5 mg/kg averaged 108%, but showed high variability, with a relative standard deviation of 17%. The technique represents an alternative to technologies such as surface plasmon resonance, with an advantage in that the flow of extracts through small capillaries is not required.