Ochratoxin A (OTA) is a major concern for public health and the rapid detection of trace OTA in food is always a challenge. To minimize OTA exposure to consumers, a nanobody (Nb)-mediated förster resonance energy transfer (FRET)-based immunosensor using quantum dots (Nb-FRET immunosensor) was proposed for ultrasensitive, single-step and competitive detection of OTA in agro-products at present work. QDs of two sizes were covalently labeled with OTA and Nb, acting as the energy donor and acceptor, respectively. The free OTA competed with the donor to bind to acceptor, thus the FRET efficiency increased with the decrease of OTA concentration. The single-step assay could be finished in 5 min with a limit of detection of 5 pg/mL, which was attributed to the small size of Nb for shortening the effective FRET distance and improving the FRET efficiency. The Nb-FRET immunosensor exhibited high selectivity for OTA. Moreover, acceptable accuracy and precision were obtained in the analysis of cereals and confirmed by the liquid chromatography-tandem mass spectrometry. Thus the developed Nb-FRET immunosensor was demonstrated to be an efficient tool for ultrasensitive and rapid detection of OTA in cereals and provides a detection model for other toxic small molecules in food and environment.