Z-Asp-Val-Tyr-NH2, a precursor tripeptide of thymopentin(TP-5), was successfully synthesized by a combination of chemical and enzyme methods. Firstly, the precursor dipeptide Val-Tyr-NH2 was synthesized by a novel chemical method in four steps including the preparation of NCA-Val, the esterification of L-tyrosine and the ammonolysis of L-tyrosine ethyl ester, and the formation of Val-Tyr-NH2 from NCA-Val and L-Tyr-NH2. Secondly, a thermolysin, thermoase PC 10F was used to catalyze the formation of Z-Asp-Val-Tyr-NH2 in an aqueous/organic solvent diphase system with Z-Asp-OH and Val-Ty-NH2 as the substrates under thermodynamic control. The optimum conditions were pH=6.5, 40 ℃, thermoase 100 mg, in 2-(N-morpholino) ethanesulfonic acid MES/NaOH(containing 5 mmol/L CaCl2) of n-butanol system(15/85, volume ratio) for 20 h in a maximum yield of 27.02%.