Abstract: Different column chromatography materials were selected to separate and enrich flavonoids from Apocynum venetum tea,and their antioxidant activity in vitro and anti-fatigue effect in vivo was evaluated. The results showed that polyamide resin had the best enrichment effect on flavonoids from Apocynum venetum tea,with the adsorption rate of 65.08% and the desorption rate of 41.27%. The antioxidant experiment in vitro showed that flavonoids in Apocynum venetum tea had strong scavenging ability on ABTS⁺·,DPPH·,·OH free radicals. When the concentration was 0.20 mg/mL,the DPPH clearance rate was 98.5%±0.18%,which was better than that of the positive control group V_C at the same concentration,indicating good antioxidant ability. The anti-fatigue test in vivo showed that flavonoids of Apocynum venetum tea could prolong the exhaustive swimming time of mice,increase the liver glycogen content,reduce serum urea nitrogen content and enhance the activity of antioxidant enzymes in vivo,all of which pointed out that flavonoids in Apocynum venetum tea had well anti-fatigue effect.