In this study, the contents of rutin and isoquercetin in flavonoids of Apocynumvenetum tea was determined by high performance liquid chromatography (HPLC), and the antioxidant effect in vitro was evaluated. A good separation of rutin and isoquercetin from flavonoids of Apocynumvenetum tea was achieved by vacuum heating reflux combined with macroporous adsorption. Rutin and isoquercetin were separated effectively in the extract of Apocynum venetum teaflavonoids. The results showed good linear relationship, the range of rutin was 0.453 1~9.062 5 μg (r=0.999 55) and that of isoquercetin ranged from 0.258 3 to 5.166 5 μg (r=0.999 65). The recoveries of rutin and isoquercetin were 99.91% and 100.05% (n=9). The accuracy RSD of this method were respectively 0.11% and 0.10% (n=5). The contents of rutin and isoquercetin in Apocynum venetum tea flavonoids were 0.059 4, 0.673 4 mg/g respectively. Moreover, the antioxidant experimental results showed that Apocynum venetum tea flavonoids had higher scavenging abilities of 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydroxyl radicals than V_C positive control. It can be found that rutin and isoquercitrinwere the main components of flavonoids in Apocynumvenetum tea. The flavonoids in Apocynumvenetum tea had good antioxidant activitiesin vitro.