Abstract Strong chemiluminescence was observed when cerium(IV) reacted with rhodamine 6G in sulfuric acid medium in the presence of puerarin. This phenomenon has been utilized to design a sensitive and selective flow injection-chemiluminescence method for the determination of puerarin. Under the optimum conditions, the proposed procedure has a linear range between 1.3 × 10 −9 and 8.0 × 10 −7 g/mL, with a detection limit of 8.4 × 10 −10 g/mL puerarin and a relative standard deviation of 1.86% ( n = 11) at 5.0 × 10 −8 g/mL puerarin. The method was successfully applied to the determination of puerarin in pharmaceutical preparations. The mechanism of this chemiluminescence reaction has been proposed.