Acid orange II (AO2) is a synthetic azo dye commonly used illegally in food production and processing. The development of a fast and effective analytical method is crucial for controlling the unlawful use of AO2 in food, especially in seasoning. Here, an antibody specific for AO2 was generated, with 1.01 ng/mL IC50 and minimal cross-reactivity with other analogs (<1 %, including sunset yellow, tartrazine, para red, chromotrope FB, Sudan Ⅰ, rhodamine B, 2-naphthol, amaranth, ponceau 4 R and allura red AC). Furthermore, a highly sensitive quantum dot beads-based fluorescence immunochromatography assay (QBs-FICA) was established for AO2 determination. The assay had a working range of 23–287 μg/kg with a 11 μg/kg limit of detection (LOD) in chili powder and hotpot seasoning samples. Using spiked samples, the results of QBs-FICA were found to be consistent with those of liquid chromatography-tandem mass spectrometry. This indicates that QBs-FICA is a reliable, rapid, and portable tool for testing of AO2.