In order to obtain the basic data for synthetic studies of bioactive peptide using enzyme, Kyotorphin(analgesic peptide) derivative was synthesized from Ac-Tyr-OH and $Arg-NH_2$ by $alpha-chymotrysin$ in two phase system(organic phase and aqueous phase). In effect of organic solvent on Kyotorphin derivative synthesis from Ac-Tyr-OH(10 mM) and $Arg-NH_2$ (20 mM), the synthesis in ethyl acetate system of organic solvents was higher than those in other organic solvents (n-butanol, n-hexane, dichloromethane and chloroform). The optimal conditions for the synthesis are as follows: enzyme conc., 10 ${mu}M;$ reaction pH, 7.0; reaction temp., $35^{circ}C$ ; the ratio of organic phase volume/aqueous phase volume $(alpha)$, 15. Under the optimal conditions, the yield was 70.2%, and the reaction achieved to equilibrium after 24 hrs.