A novel modification of the method ordinarily used for preparing surfactant-enzyme complexes that can be employed in organic media was developed utilizing a water-in-oil (W/O) emulsion. The modified method entails coating a selected enzyme with an appropriate surfactant by creating a W/O emulsion which encapsulates the enzyme in organic media. This method of preparation involves only a few simple stages, while it offers the advantages of enhanced enzymatic activity in reaction media and eliminates enzyme loss. Using a surfactant-chymotrypsin (CT) complex, transesterification of an amino acid derivative was carried out in an organic solvent and the optimal conditions for achieving a suitable surfactant-CT complex were investigated. The kind of surfactant coating and the aqueous pH at the time of complex preparation were key factors in obtaining a high level of enzymatic activity in organic media. The enzymatic activity of the CT complex was significantly enhanced by coating it with a nonionic surfactant. A pH of 9 was most appropriate for the preparation of the coated CT complex, although this pH value is a little higher than that obtainable in ordinary enzymatic reactions in aqueous media optimized at pH 7.8. The CT complex retained a high level of enzymatic activity when stored for one month at 277 K. This modified method of surfactant-enzyme complex preparation will facilitate the use of enzymes in organic media as new biocatalysts.