A novel preparation method for surfactant-enzyme complexes applicable in organic media is developed utilizing a water-in-oil (W/O) emulsion. This preparation method entails simple stages while it offers the advantages of enhanced enzymatic activity in the reacting media and eliminates enzyme loss. Using the surfactant-chymotrypsin complex, peptide synthesis with amino acid derivatives was conducted in tetrahydrofuran. The surfactant-chymotrypsin complex indicates higher activity than native chymotrypsin. This modified method will facilitate the usage of enzymes in organic media as new biocatalysts.