Abstract: The crude alcoholic extract of Brassica napus L. bee pollen was treated by solvent extraction and macroporous resin, and α-glucosidase inhibition test on the depurated flavonoids in vitro was carried out. The components which had the strongest inhibitory effect on α-glucosidase were analyzed by IR and LC-MS.Results showed that the flavonoids played a vital role in inhibiting α-glycosidase enzymes. The purity of PEFS-3 which was made by AB-8 macroporous resin was 68.77%, and its IC50 was 72.16μg/m L, far less than the IC50 of acarbose (1124.86μg/m L) . Based on IR and LC-MS, five main components were identified in PEFS-3, and four kinds among them were flavonoid aglycones:Quercetin-3-O-β-D-pyranoid glucose base- (1→2) -β-Dpyranoid glycosidase, Kaempferol-3, 4 '-double-O-β-D-pyranoid glycosidase, Isohtamnetin-3-O-β-D-pyranoid glucose base- (1→2) -β-D-pyranoid glycosidase, Kaempferol-3-O-β-D-pyranoid glucose base- (1→2) -β-D-pyranoid glycosidase, and the fifth one was inferred to be sec-O-glucosylhamaudol or flavanol, its specific structure still need further confirmation.