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 when materials bind to the tip of the fiber. 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. In this report, a deoxynivalenol-bovine serum albumin (DON-BSA) conjugate was non-covalently immobilized to the surface of aminopropylsilane sensors and the change in interference pattern resulting from the binding of DON-specific antibodies was measured. The basis for the assay was the competition between DON and the immobilized DON-BSA for binding to limited amounts of antibody. The technique was used to measure DON in extracts of 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. When samples were tested with sensors that had not been conditioned to remove loosely attached DON-BSA, the recoveries at five spiking levels over the range from 0.2 to 5 mg/kg averaged 108.8% [relative standard deviation (RSD) 16.0%]. Using sensors that had been conditioned lowered the average recovery (101.4%) and improved the RSD (13.2%). This suggests that conditioning the sensors helped reduce a bias in the assay towards overestimation. These results, and the ease with which assays can be conducted, suggest further exploration of this technology for detection of mycotoxins is warranted.