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Profenofos (PFF) is a commonly used organophosphorus insecticide that requires strict monitoring due to its potential environmental, ecological, and human health risks originating from residues in soil and water systems, as well as accumulation in crops. In this study, a novel monoclonal antibody (mAb) targeting PFF was prepared for the first time and investigated the recognition mechanism through molecular simulation. Subsequently, a mAb-based colloidal gold immunochromatographic assay (GICA) was developed for the rapid screening of PFF in fruit and vegetable samples. The mAb exhibited an IC 50 value of 12.9 ng/mL, and limit of detection (LOD) of 4.6 ng/mL, respectively in indirect competitive immunosorbent enzyme-linked immunosorbent assay (ic-ELISA). After optimization, the developed GICA exhibited a visual limit of detection (vLOD) of 20 ng/mL and a quantitative of detection (qLOD) of 5.2 ng/mL, with a linear range from 10.0 to 83.8 ng/mL. Good correlation was observed between the results of GICA and standard Gas Chromatography-Tandem Mass Spectrometry (GC-MS/MS) in matrix and recovery test. The developed GICA can be used for rapid sample detection within 15 minutes, which is an excellent tool for screening PFF in foods and environmental samples., Competing Interests: Declaration of Competing Interest ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2024. Published by Elsevier Ltd.)