RGD tripeptide as the cellular adhesion factor was synthesized by a combination of chemical and enzymatic methods in this study. First of all, Gly-Asp diamide was synthesized by a novel chemical method in three steps including preparation of L-aspartic acid dimethyl ester, chloroacetylation of L-aspartic acid dimethyl ester and ammonolysis of chloroacetyl L-aspartic acid dimethyl ester. Secondly, Porcine Pancreas Lipase (PPL) was used to catalyze the synthesis of Benzyl-Arg-Gly-Asp diamide in water-organic cosolvents systems. The reaction condition was optimized by examining the main factors affecting the yield of the tripeptide. The optimal reaction condition was set up as pH7.7, 15 degreesC in 50% DMF for 8 h with the maximum yield of 76.4%. It was also found that 50% DMSO was another alternative with the tripeptide yield of 71.7%.