A new kind of weak cation exchange (WCX) stationary phase for high performance liquid chromatography (HPLC) was synthesized by the chemical modification of 5.0 microm macroporous monodisperse hydrophilic poly (glycidylmethacrylate-co-ethylenedimethacrylate) (P(GMA/EDMA)) beads. The stationary phase was evaluated in detail to determine its separability, hydrophilicity, reproducibility and stability. It was found that the column exhibited excellent properties on the protein separation, reproducibility and stability. Four proteins were separated with linear gradient elution on the synthesized WCX stationary phase in 6 min. The highest dynamics protein loading capacity of the WCX packing for lysozyme (Lys) was 29.86 mg/g. The WCX packing was used for the fast separation and purification of protamine from carp with only one step. The purity of the obtained protamine detected by reversed-phase HPLC was 99.2%. Therefore satisfactory results have been obtained. Compared with the Shodex IEC SP-825 strong cation exchange column for the purification of protamine, the purity of protamine obtained from this column was almost same.