A novel molecularly imprinted polymer (MIP) selective for tryptophan (Trp) was described where polymerization was performed in aqueous media. Three kinds of molecularly imprinted polymers were prepared with surface molecular imprinting technique on functionalized silica gel (F-silica gel). MIPs prepared using bonded beta-cyclodextrin (beta-CD) and acrylamide (AA), either separately or in combination have shown various recognition properties. The results of adsorption experiments indicated that the selectivity of MIP, which was synthesized with bonded beta-CD and AA [MIP(1)], was superior to those obtained with AA [MIP(2)] or bonded beta-CD [MIP(3)]. In addition, the high-performance liquid chromatography (HPLC) column packed with MIP(1) could not only separate Trp from other aromatic amino acids, but also separate the template from its enantiomer in aqueous mobile phase. This study developed a new method for chiral amino acid separation and purification.