An aminocarboxy aspartic acid-bonded silica (Asp-Silica) stationary phase was synthesized using L-aspartic acid as ligand and silica gel as matrix. The standard protein mixtures were separated with prepared chromatographic column. The effects of solution pH, salt concentration and metal ion on the retention of proteins were examined, and also compared with traditional iminodiacetic acid-bonded silica (IDA-Silica) column. The results show that Asp-Silica column exhibited an excellent separation performance for proteins. The retention of proteins on Asp-Silica stationary phase was consistent with electrostatic characteristic of cation-exchange. The stationary phase displayed typical metal chelate property after fixing copper ion (II) on Asp-Silica. Under competitive eluting condition, protein mixtures were effectively isolated. Asp ligand showed better ion-exchange and metal chelating properties as compared with IDA ligand.