Fenamiphos (FENA) is an organophosphorus insecticide, and its residues in fruits, vegetables, and the environment have raised concerns. Therefore, it is very important to develop a simple, rapid, and accurate method for FENA detection. In this study, a novel FENA hapten was designed and predicted based on computer-aided simulation technology, and high-performance anti-FENA monoclonal antibodies were screened using a matrix effect-enhanced screening method, with a half-maximal inhibitory concentration of 1.269 ng/mL. Furthermore, a colloidal gold immunochromatographic assay (ICA) was established for the detection of FENA, with calculated limits of detection of 1.030 μg/kg, 0.515 μg/kg, 0.250 ng/mL, and 0.405 μg/kg in oranges, cowpeas, river water, and soil. Spiked recovery experiments and real sample validation showed that the detection results from the ICA and LC-MS/MS were consistent, with a CV <
10 %, indicating that the ICA had good accuracy and stability and shows promise as a rapid screening method for FENA in fruits, vegetables, and the environment.