The invention discloses a glucan-poly(hydroxyethyl methacrylate) based continuous bed separation medium used in the field of biochemical separation and a preparation method thereof. The pore size range of the separation medium is 1-200 mu m, the porosity is 83-96%, and the adsorption capacity for a model protein such as bovine serum albumin can be up to 5-9 mg/mL cryogel. According to the invention, a matrix scaffold of the provided glucan-poly(hydroxyethyl methacrylate) based continuous bed cryogel separation medium is a copolymerization material of glucan derivatives and hydroxyethyl methacrylate, has favorable biocompatibility and safety and can be reutilized; and functional groups thereof are anion-exchanging and hydrophobic functional dual-mode groups and have excellent separation performance. The preparation method of the glucan-poly(hydroxyethyl methacrylate) based continuous bed cryogel separation medium is simple and easy to implement and easy to realize large-scale preparation, and has wide application prospects in the field of biochemical separation.