The present invention relates to anti-human NKX3.1 monoclonal antibody and its preparation method and application. The invention provides an anti-human NKX3.1 monoclonal antibody, and further provides the amino acid sequences of the variable regions of its heavy chain and light chain. On this basis, the antibody can be prepared by conventional genetic engineering methods. Compared with the traditional monoclonal antibody preparation method, the anti-human NKX3.1 recombinant antibody prepared by the genetic engineering method of the present invention has the advantages of known sequence, stable antibody properties, good repeatability, etc., and can specifically recognize human NKX3.1 protein , can be used for IHC immunohistochemical detection. And the standardized antibody production process avoids the risk factors that appear in the traditional monoclonal antibody production and storage process. Furthermore, on the basis of the amino acid sequence of the antibody provided by the present invention, one or more amino acid additions, deletions, substitutions, etc., can also be modified to obtain active fragments or conservative variants, in order to further improve the specificity and Affinity lays the groundwork.