Objective To produce anti‐19‐Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics. Methods Hybridomas were prepared by fusing NS0 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 hybridoma cell lines, named NT‐1, NT‐2, NT‐3, NT‐4, and NT‐5, were identified and their corresponding mAbs were of the IgG 1 isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7×10 9 L/mol. The titers and IC 50 values of purified ascite fluids were in the range of (0.64-2.56)×10 5 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 (<0.01%) with other steroids was observed. Conclusion The establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals. Objective To produce anti-19-Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics. Methods Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB / c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cells Results Five hybridoma cell lines, named NT-1, NT-2, NT, were used to purify the NT mAb, and the Batty saturation method was used to determine the affinity constant (Kaff) -3, NT-4, and NT-5, were identified and their corresponding mAbs were of the IgG 1 isotype with a light chain. The Kaffs of all mAbs were between 2.6 and 4.7 × 10 9 L / mol. The titers and IC 50 values ​​of purified ascites were in the range of (0.64-2.56) × 10 5 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. Negli gible cross-reactivity (<0.01%) with other steroids was observed. Conclusion The establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.