Dicamba, a traditional highly effective and low toxicity herbicide, has gained new life with the development of dicamba-tolerant transgenic crops in recent years. However, dicamba is highly volatile and therefore easy to cause drift damage to sensitive crops. The development of efficient and sensitive detection methods is essential for monitoring of trace dicamba in the environment. Nanobody-based immunoassay plays an important role in on-site detection of pesticides. However, now rapid and sensitive immunoassay methods based on nanobody for dicamba detection were lacking. In this study, the nanobodies specifically recognizing dicamba were successfully obtained by immunising camels and phage display library construction, and then an indirect competitive immunoassay based on Nb-242 was constructed with IC of 0.93 μg/mL and a linear range of 0.11-8.01 μg/mL. Nb-242 had good specificity with no cross-reactivities against the dicamba analogs other than 2,3,6-trichlorobenzoic acid and the developed immnoassay had a good correlation with the standard HPLC in the spike-recovery studies. Finally, the key amino acid Ala 123, Tyr 55, Tyr 59 and Arg 72 of Nb-242 that specifically recognizing and binding with dicamba were identified by homologous modeling and molecular docking, laying an important foundation for further structural modification of Nb-242. This study has important guiding significance for constructing immunoassay method of dicamba based on nanobody and provides a sensitive, specific, and reliable detection method that is suitable for the detection of dicamba in the environment.