Residues of diquat (DQ), a bipyridyl herbicide, can pose a threat to human health. In this study, we designed two semi-antigens and obtained a highly specific monoclonal antibody (mAb) against DQ. The half maximal inhibitory concentration of mAb was 0.63 ng mL⁻¹ with a limit of detection of 0.14 ng mL⁻¹. Using this specific mAb, we developed an immunochromatographic assay with a cut-off value of 0.1 mg kg⁻¹ in fruit samples, which was consistent with the HPLC-MS results, confirming that the test strips can be used in real samples. Our developed test strip can be used for screening DQ in food samples. Compared to instrumental methods, test strips offer several advantages.