A lateral flow immunochromatographic test strip was developed for on-site rapid and sensitive detection of Hexoestrol (HES) residues in fish samples with colloidal gold labeled the anti-HES monoclonal antibody (mAb). The strip is composed of a sample pad, a conjugate reagent pad, an absorbent pad, and a test membrane containing a control line and a test line. The sensitivity (half inhibitory concentration, IC50) of the strip in the detection of fish extract samples was confirmed to be 1.86 μg/kg, and the limit detection (LOD) value was 0.62 μg/kg. For intra-assay and inter-assay reproducibility, recoveries of HES spiked samples were ranged from 86.3% to 92.3% and 85.8% to 93.4%, coefficients of variation were 2.91-4.64% and 4.24-5.17% respectively. High-performance liquid chromatography (HPLC) was employed to confirm the performance of the strip. The strip test only took less than 10 minutes, and thus provides a repaid method for on-site detection of HES residues.