The invention relates to a separation medium, a preparation method and applications thereof, wherein the separation medium has a structure represented by a formula I, SP is a medium, R is a spacer arm, and rProtein A-cys is a recombinant protein A. The new separation medium provided by the invention has advantages of dynamic binding capacity of 50 mg / mL, high separation efficiency, good separationeffect and low cost, and is suitable for industrial large-scale separation and purification of antibodies and Fc fusion proteins.