Abstract: Phenamacril is a novel fungicide invented independently for the control of wheat head scab in China. No similar product is available at home or abroad, and no immunoassay method has been reported for its’ residue detection. In this study, a murine-derived specific monoclonal antibody (mAb) against phenamacril was prepared through design and synthesis of a hapten. Thereafter, a colloidal gold immunochromatographic assay was established using nanogold labeling. The characterization results revealed that the subtype of achieved mAb was IgG1. The results of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) showed that in the linear range of 38.72 to 438.08 ng/mL, the detection sensitivity of the monoclonal antibody to phenamacril was 38.72 ng/mL at a 20% inhibition concentration (IC₂₀), and 108.42 ng/mL at a 50% inhibition concentration (IC₅₀). In addition, the ic-ELISA showed the cross-reactivities (CRs) of the mAb to structural analogues (azoxystrobin, pyraclostrobin, fluopimomide, and picoxystrobin) of the fungicides were less than 0.01%, which exhibited high specificity of the mAb for detection of phenamacril. The minimum limit of detection (LOD) reached to 50 ng/mL of colloidal gold immunochromatographic strip with naked eye observation, and the detection could be completed within 15 minutes. The immunochromatographic assay also showed negligible cross-reactivities to methoxyacrylate fungicides. Method validation was conducted with phenamacril spiked wheat flour samples, and the results of colloidal gold immunochromatographic strips were consistent with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. In conclusion, the developed immunoassay can achieve rapid qualitative as well as semi-quantitative analysis of phenamacril residues in samples.