Abstract: A novel boric acid-affinity chromatographic packings was prepared by using 3.0 μm non-porous monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads (P(GMA/EDMA) as matrix and M-aminophenylboric acid (APBA) as ligand. The specific effects of antibiotics, such as norvancomycin, amikacin, isopamicin and gentamicin, with boric acid ligands, as well as the effects of salt ion concentration and pH in mobile phase on the retention of antibiotics were investigated. The results indicated that antibiotics with o-hydroxyl groups and borate groups had affinity specificity, and antibiotics with the different number of o-hydroxyl groups in structure showed different retention ability. Furthermore, the norvancomycin was purified by boric acid affinity column and the purified solution was determined by reversed-phase column, proving that the developed boric acid affinity chromatographic column in this study was capable of purifying norvancomycin.