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BACKGROUND: Nitroxynil (NIT) is a veterinary drug against hepatic fluke disease for food‐producing cattle and sheep. NIT has a long half‐life time in animals since it is highly bound to plasma protein. Therefore NIT possibly remains in animal edible tissues or milk due to drug abuse. In this study, a specific murine monoclonal antibody (mAb) against NIT was prepared and an immunochromatographic strip assay based on the mAb was developed for screening NIT in milk. RESULTS: The affinity constant of the anti‐NIT mAb was 2.93 × 1010 and the anti‐NIT mAb had almost no cross‐reactivity with other analogs, so that it showed good specificity. The cutoff value of this test strip was considered to be 50 ng mL−1 by the naked eye. When detected by the strip reader, the half maximum inhibition concentration (IC50) of the immunoassay strip was calculated to be 5.716 ng mL−1 and the limit of detection (LOD) was 1.146 ng mL−1. Intra‐assay recoveries from 88.80 to 97.13% were obtained, with the highest coefficient of variation (CV) at 9.01%; inter‐assay recoveries ranged from 84.60 to 106.87%, with the highest CV at 9.93%. CONCLUSION: The operative procedure of the proposed method can be completed within 10 min. The strip developed in this study was a practical tool for rapid semiquantitative and quantitative detection of NIT in milk. This study suggested great potential for analytically monitoring NIT in other food samples. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]