An on-line HPLC-diode array detection-flow injection chemiluminescence (HPLC-DAD-FICL) method was applied to estimate the difference of Puerariae lobatae and Puerariae thomsonii. Their chemical and active profiles could be obtained by HPLC-DAD-FICL in one run. Seventeen compounds in two species were tentatively identified by HPLC-electrospray ionization-MS (HPLC-ESI-MS) method. The main antioxidants were rapidly screened by active fingerprints coupled with MS data. Similarity and Hierarchical clustering analysis (HCA) were used to distinguish different samples. The results suggested that the chemical fingerprints of 16 batches of samples were similar by similarity evaluation, while HCA could discriminate the two species. The active fingerprints of Puerariae lobatae and Puerariae thomsonii were significantly different. More antioxidants were found in Puerariae lobatae than in Puerariae thomsonii. Main antioxidants, including 3'-hydroxypuerarin, genistein 8-C-glycoside-xyloside, puerarin, 6″-O-xylosylpuerarin, mirificin and daidzein in two species, may be reasonable markers for the discrimination of the two species. The integrated fingerprint based on the chemical and active characteristics may provide an objective quality evaluation for Puerariae lobatae and Puerariae thomsonii.