The invention provides preparation methods and application of pendimethalin hapten and antigen. The pendimethalin hapten is characterized by being prepared by taking N-(1-ethylpropyl)-3,4-dimethylaniline and 4-ethyl bromobutyrate to react to generate alkylated N-(1-ethylpropyl)-3,4-dimethylaniline, taking the alkylated N-(1-ethylpropyl)-3,4-dimethylaniline and concentrated nitric acid to react togenerate nitro-alkylated N-(1-ethylpropyl)-3,4-dimethylaniline, and then reacting with potassium hydroxide; the pendimethalin antigen is prepared by coupling the pendimethalin hapten and carrier protein. The antigen prepared by the invention has a specific pendimethalin antigen determinant, so that a high-specificity pendimethalin monoclonal antibody is possibly screened. The produced antibody hashigh specificity and high sensitivity and can be used for establishing an enzyme-linked immunosorbent assay method and a colloidal gold test strip determination method, so that rapid detection of pendimethalin in tobaccos and foods is realized.