A polyethyleneimine (PEI)-functionalized polymer substrate polar stationary phase was prepared for hydrophilic interaction chromatography (HILIC) by grafting PEI onto poly(styrene-divinylbenzene) (PS-DVB) microspheres. The phase shows a U-shape retention profile and it exhibits typical hydrophilic characteristic when the organic solvent fraction in the mobile phase is60%. Hydrogen bonding, anion exchange, and hydrophobic interaction are involved in the retention mechanism. Good separation and unique selectivity for acidic, basic and neutral polar analytes were achieved. It showed extremely low column bleed (comparable to that of blank) under gradient elution mode (even to 50% fraction of water) and wide pH tollerance range (at least 1-13).