Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, EM-1), an effective analgesic, was efficiently synthesized by a combination of enzymatic and chemical methods. Peptide Boc-Trp-Phe-NH2 was synthesized with a high yield of 97.1% by the solvent-stable protease WQ9-2 in a 20% methanol medium. The maximum concentration (141 g L−1) of Boc-Trp-Phe-NH2 was obtained with an economical molar ratio of the substrate of 1:1. The products crystallized and separated from the substrates without purification, followed by the removal of the Boc group with trifluoroacetic acid to generate Trp-Phe-NH2. Using the efficient mixed carbonic anhydride method, Boc-Tyr-Pro-OH was synthesized chemically. The tetrapeptide Boc-Tyr-Pro-Trp-Phe-NH2 was synthesized with a yield of 84.5% by another organic solvent-tolerant protease, PT121, from Boc-Tyr-Pro-OH and Trp-Phe-NH2 in an organic–aqueous biphasic system and was extracted with ethyl acetate, shifting the equilibrium of the synthesis. EM-1 was obtained by removal of the Boc group from Boc-Tyr-Pro-Trp-Phe-NH2 in a high yield of 91% due to the free protection of the side-chain of the Tyr phenolic hydroxy group. After a one-step purification in the final step by using high speed countercurrent chromatography (HSCCC), EM-1 was obtained with a purity greater than 99.8%. The chemo-enzymatic synthesis of EM-1 proved to be efficient, productive, with minimal side-chain protection and simple purification, thus greening the synthesis of the peptide.