To extract and isolate the flavonoids from Oxytropis falcata (Of)，and screen bioactive flavonoids on human lung adenocarcinoma A549 cells in vitro.Methods:Crude extraction was extracted from Of by ethyl acetate.Ethyl acetate extraction was further separated by polyamide column chromatography with gradient elution and purified by medium-pressure liquid chromatography to obtain the flavonoids; High performance liquid chromatography (HPLC)，mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) were used to analyze and elucidate the structure of the obtained flavonoids.CCK-8 analysis was used to detect the inhibited viability of flavonoids on human lung adenocarcinoma A549 cells in vitro.Results:There were 2 flavonoids isolated from Of ethyl acetate fraction，and were identified as 7-hydroxy dihydroflavone and 2'，4'-dihydroxy dihydrochalcone.Purity of them were both greater than 98% by HPLC analyses; The results of CCK-8 showed that 7-hydroxy dihydroflavone and 2'，4'-dihydroxy dihydrochalcone could significantly inhibit the viability of A549 cells in a dose dependent manner.The IC50 values were 137.6 μg/mL and 63.83 μg/mL at 48 h，respectively.Conclusion:The 2 flavonoids have effective bioactivities on inhibiting proliferation of human lung adenocarcinoma A549 cells，which could provide the experimental basis for the further studies on anti-lung cancer mechanism.Polyamide column chromatography is suitable for enrichment and isolation of flavonoids.Polyamide column chromatography combined with medium pressure liquid chromatography is simple and easy to prepare various flavonoids.Thus，the method could provide a reference for obtaining flavonoids from Chinese medicines.