A noncompetitive and homogeneous fluorescence resonance energy transfer (FRET) immunoassay was developed using a nanobody (Nb) for highly sensitive and simultaneous detection of ochratoxin A (OTA) and ochratoxin B (OTB). The promoted intrinsic fluorescence (λex: 280 nm) of tryptophan residues (donor) in Nb can excite the fluorescence of OTA and OTB (acceptor) for detection (λem: 430 nm). Using optimal conditions, the limits of detection of the Nb-based FRET immunoassay were 0.06 and 0.12 ng/mL for OTA and OTB, respectively. Minimal cross reactivity was detected for several analogues of OTA and OTB as well as nonspecific proteins and antibodies. Acceptable accuracy and precision were obtained in the spike and recovery study, and the results correlated well with those by HPLC. These results demonstrated that the developed method could be a useful tool for noncompetitive, homogeneous, and simultaneous detection of OTA and OTB as well as other environmental analytes with similar fluorescence properties.