With the rapid development of antibody engineering,single chain fragment of variable(scFv)-alkaline phosphatase(AP)fusions is considered an attractive alternative for simple and rapid immunoassay analysis,avoiding the chemical conjugation of enzymes to antibodies and the use of a second antibody.However,scFvs are often observed to have poor solubility and stability which require additional genetic engineering to improve.As an alternative to conventional antibody,nanobody(Nb)which is derived from the variable region of heavy chain antibody existing in camelids and sharks,is more attractive than scFv.Nb(15 kDa)is smaller than scFv(75 kDa)and exhibits high solubility and the ability to refold properly after denaturation by heat or chemical means.In our previous work,we reported on the panning of binders specific for ochratoxin A from an immunized alpaca Nb library,and sixteen binders with four different amino acid sequences were selected.In this study,one binder with the highest sensitivity in phage ELISA was used to prepare the Nb-AP construct for expression.The purified fusion protein was used to develop a sensitive fluorescence enzyme immunoassay(FEIA)for OTA detection in cereal.The 50%inhibitory concentration and the detection limit of the dc-FEIA were 0.133 ng/mL and 0.038 ng/mL,respectively,with a linear range of 0.141-1.065 ng/mL.Validation results from FEIA and LC-MS/MS were in good agreement with each other,indicating the reliability of this assay for application in the rapid screening of OTA in cereal samples.