The phenomenon of fluorescence quenching of an antibody by a specific ligand was applied in developing a technique for detection of mycotoxins, such as aflatoxin B₁ (AFB₁), ochratoxin A, and zearalenone. Studies showed that the intrinsic fluorescence of tryptophan (Trp) residues in antibodies, promoted by excitation at 280 nm, is quenched upon binding of specific mycotoxin ligands. Fluorescence quenching in FRET system takes place in these systems as a consequence of the overlap of the emission spectra of antibody donors with the absorption spectra of the mycotoxins. Further studies focusing on the detection of AFB₁ revealed that the Fab fragment, the variable region of the antibody where specific binding of AFB₁ occurs, can be utilized to increase the sensitivity of the detection system. The results demonstrate that fluorescence of the Fab fragment is almost completely quenched by AFB₁ whereas emission from intact anti-AFB₁ is only partially quenched by this mycotoxin. The limits of detection (LODs) were found to be 0.85 and 0.09 ng mL⁻¹ for assays using the intact antibody and the Fab fragment, respectively, corresponding to a 10-fold enhancement. A practical application of the Fab fragment based assay system was demonstrated by its use in the detection of AFB₁ in spiked barley grain samples. The observations made in this effort show that the new, label-free, non-competitive, and homogeneous FRET immunoassay strategy, which requires a simple sample preparation procedure, serves as a powerful tool for the rapid and sensitive quantitative determination of organic substances such as mycotoxin.