Poly (glycidylmethacrylate-divinylbenzene) microspheres were prepared by the two-staged swelling and polymerization method and applied to prepare anion exchange stationary phases. Methylamine, dimethylamine, trimethylamine, diethylamine and triethylamine were selected to prepare the quaternary ammonium groups of anion exchangers, respectively. The diameters and surface characteristics of microspheres were measured by scanning electron microscope and nitrogen adsorption-desorption measurements. The anion exchangers were characterized by Fourier transform infrared spectrum, elemental analysis and breakthrough curve methods. The chromatographic performances of anion exchangers were illustrated by separating conventional anions, organic weak acids and carbohydrates. The results indicated that the anion exchange capacities were controllable by changing either the content of glycidylmethacrylate in microspheres or the number of bonded quaternary ammonium layer. Meanwhile, the substituents of quaternary ammonium groups greatly influenced the separation properties of anion exchangers. Finally, the three-layer methylamine-quaternized anion exchanger was successfully applied for the determination of fluoride in tea sample. The content of fluoride was detected to be 0.13mgg−1 without the interference of acetate and formate.