The separation of baicalein and baicalin, two major flavonoids in Scutellaria, by using collagen fiber adsorbent (CFA), was investigated. Static adsorption and adsorption kinetics indicated that baicalin had stronger hydrogen-bond interaction with CFA in high ethanol concentration solution, while baicalein presented stronger hydrophobic interaction with CFA in low ethanol concentration solution. In CFA column chromatography separation, baicalein and baicalin could be well separated by stepwise elution of 90% and 70% aqueous ethanol solutions, and their recoveries were 83.42% and 94.31%, respectively. The maximum loading mass for separating baicalein and baicalin on a CFA column was higher than 90 mg/g CFA, which is much more than the loading mass on a D101 macroporous resin column. Applications repeated 10 times indicated that the CFA column has satisfactory reusability. Furthermore, the separation of baicalein and baicalin from Scutellaria extracts using CFA was investigated. The purity of baicalein and baicalin obtained was both higher than 98%.