With the development of colloid interface and enzyme technologies, enzyme-containing reversed micellar system has been receiving much attention in bioseparation and bioconversion. Because of its high efficiency, it has brought new opportunities for the development of molecular biotechnology. Reversed micelles represent nano-sized aqueous droplets stabilized by surfactant amphiphiles inside the bulk organic solvents. The entrapped enzymes have enhanced activities under those conditions as suited in the lipid bilayers of biological membranes. The fundamentals of enzyme-containing reversed micellar system are described in this paper, with special emphasis on the effects of surfactants varying in concentrations and structures. The latest study progress on the surfactants application in enzyme-containing reversed micelles is reviewed. The introduction of novel functional surfactants in micellar enzymology and their future development are also discussed.