Quenchbody (Q-body) is a novel fluorescent biosensor based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. In order to develop a method using Q-body for the quantitative determination of deoxynivalenol (DON), a trichothecene mycotoxin produced by some Fusarium species, anti-DON Q-body was synthesized from the sequence information of a monoclonal antibody specific to DON. When the purified anti-DON Q-body was mixed with DON, a dose-dependent increase in the fluorescence intensity was observed and the detection range was between 0.0003 and 3 mg L(-1). The coefficients of variation were 7.9% at 0.003 mg L(-1), 5.0% at 0.03 mg L(-1) and 13.7% at 0.3 mg L(-1), respectively. The limit of detection was 0.006 mg L(-1) for DON in wheat. The Q-body showed an antigen-dependent fluorescence enhancement even in the presence of wheat extracts. To validate the analytical method using Q-body, a spike-and-recovery experiment was performed using four spiked wheat samples. The recoveries were in the range of 94.9-100.2%. The concentrations of DON in twenty-one naturally contaminated wheat samples were quantitated by the Q-body method, LC-MS/MS and an immunochromatographic assay kit. The LC-MS/MS analysis showed that the levels of DON contamination in the samples were between 0.001 and 2.68 mg kg(-1). The concentrations of DON quantitated by LC-MS/MS were more strongly correlated with those using the Q-body method (R(2) = 0.9760) than the immunochromatographic assay kit (R(2) = 0.8824). These data indicate that the Q-body system for the determination of DON in wheat samples was successfully developed and Q-body is expected to have a range of applications in the field of food safety.