Objective To establish a new method for the determination of quercetin and kaempferol in wine by high performance liquid chromatography (HPLC) with chemiluminescence. Methods The procedure was based on the chemiluminescent enhancement by quercetin and kaempferol of the K3Fe(CN)6 system in an alkaline medium. The HPLC with post-column chemiluminescence was established for the determination of quercetin and kaempferol in wine. Results The standard curves were linear in the concentration ranges of 2.0?10-6-1.6?10-2 g/ml(r=0.999 0)for quercetin and 3.2?10-6-2.0?10-4 g/ml(r=0.999 8)for kaempfero respectively.The limits of detection were 1.4 ?10 -9 g/ml for quercetin and 3.4 ?10 -7 g/ml (S/N =3)for kaempfero respectively. The rates of recovery were 99.31% for quercetin and 99.06% for kaempferol. Conclusion The method is simple,rapid,accurate and applicable to the determination of quercetin and kaempferol in wine.