Abstract: The collagenous fiber cross-linked by glutaraldehyde was used as an adsorbent (CFA) for chromatographic separation of single glucosyl flavonoid glycosides. Genistin and baicalin with similar molecular structure were chosen as the representatives of flavonoid glycosides in the experiments is this paper. It was found that the adsorptive affinity and selectivity of CFA to genistin and baicalin could be adjusted by ethanol concentration in aqueous solution. In column chromatographic separation, where the CFA dosage was 6 g and the ratio of column height to diameter was 10:1, genistin and baicalin were well separated by stepwise elution of 100%-90%-70% ethanol aqueous solutions. The purities of the obtained genistin and baicalin were 98% and 97%, and their recovery rates were 99.35% and 96.52%, respectively.