Objective To produce anti-19-Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics. Methods Hybridomas were prepared by fusing NSO mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate (CAAP) method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant (Kaff). Results Five hybridorna cell lines, named NT-1, NT-2, NT-3, NT-4, and NT-5, were identified and their corresponding mAbs were of the IgG isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7×10 and (0.55-1.0) ng/mL, respectively. Of all the cross-reacting steroids, α-NT was the most reactive with the mAbs at 62% with NT-1 mAb and 64% with NT-2 mAb. Negligible cross-reactivity (