Abstract: Collagen fiber (CF) was previously used as packing material for the separation of flavonoids based on hydrogen bond and hydrophobic interactions. However, as for flavonoid glycosides which are rich in hydroxyls in forming hydrogen bonds, CF presented excessive adsorption capacity, causing the peak tailing and low recovery in column separation. In order to weaken the hydrogen bond interaction and improve the elution rate, carboxyls (-COOHs) were grafted on CF in this work by using glyoxylic acid (GA) to enhance the hydrophilicity of CF and the electrostatic repulsion between CF and flavonoid glycosides. Results of water contact angle, absorption time of water and isoelectric point (pI) determinations proved the successful grafting of -COOHs, and the obtained GA-modified collagen fiber (GA-CF) showed significantly enhanced hydrophilicity and decreased pI. Results of differential scanning calorimetry and scanning electron microscope determinations proved that the thermal stability and special fiber structure of collagen fiber were retained after the reacting with GA. In comparison with CF, GA-CF showed higher elution rates and separation efficiency to baicalin and myricitrin (the typical flavonoid glycosides), leading to much higher purities and recoveries. In fact, the separation performance of flavonoid glycosides on GA-CF was better than that on polyamide and comparable to Sephadex LH-20.