In this study, a rapid and highly sensitive fluorescence-linked immunosorbent assay for puerarin determination was developed by the conjugation of quantum dots with an antibody against puerarin. The linear range and detection limit of the fluorescence-linked immunosorbent assay were validated. The detection curve ( y = -1041ln( x )+5366, R ² = 0.999) was linear in the range of 7.8-125 ng/mL. The 50% inhibitory concentration determined by fluorescence-linked immunosorbent assay was 33.8 ng/mL puerarin in water. The limit of detection for PUE was 6.1 ng/mL. To our knowledge, this is the first report on the quantitative detection of a natural product using quantum dots as fluorescent markers. Furthermore, the newly developed fluorescence-linked immunosorbent assay was successfully applied to determine puerarin in several commercial Gegen Qinlian tablets, with a higher sensitivity than that of conventional enzyme-linked immunosorbent assays.