The invention belongs to the field of biotechnology, and specifically relates to the preparation and application of a nanobody that can specifically bind to an anti-deoxynivalenol antibody. Its amino acid sequence is SEQ ID NO.: 1, and it also relates to a nucleotide encoding the amino acid. The nanobody of the present invention can replace the expensive and highly toxic DON standard product, and can be used as a competitive antigen or solid-phase coated antigen in the immunological detection of DON. The nanobody has similar immune response characteristics to natural DON molecules, and the effect is good. . Compared with traditional polypeptide-based antigen mimic epitopes and IgG-based traditional anti-idiotypic antibodies, nanobodies have more stable structures, acid, alkali and high temperature resistance, and high detection sensitivity, so their immunological detection stability has been greatly improved. At the same time, the tolerance to the environment has also been improved.