Peptides are synthesized in reactions catalyzed by the protease at the optimal pH, although the enzymatic synthesis of peptides at low pH has many advantages from the view point of an industrial-scale production, such as, suppression of nonenzymatic decomposition of amino acid ester substrates and a lower requirement of NaOH for pH adjustment. We developed an extractive synthesis of peptides in an aqueous/organic biphasic system at low pH and applied it to the protease-catalyzed synthesis of N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-APM), the precursor of an artificial sweetener, aspartame. The Z-APM yield in a pure aqueous monophasic system is less than 10%, however, it was over 96% at low pH using our extractive synthesis in an aqueous/organic biphasic system although the enzyme activity was very low, The extractive synthesis at low PII has advantages, such as, suppression of substrate inhibition by decreasing the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-L-Asp) in the aqueous phase, and shifting of the chemical equilibrium towards peptide bond formation because of a high distribution of Z-L-Asp to the organic phase. We expect that our method of extractive synthesis at low pH can be applied to other reaction systems.