Vancomycin was extracted from an aqueous feed solution into a reverse micellar solution of bis(2-ethylhexyl)sulfosuccinate sodium salt in isooctane. A low pH and salt concentration of aqueous feed solutions favors forward extraction. The backward extraction efficiency of vancomycin from reverse micelles into an aqueous phase, on the other hand, increases with pH. Affinity cosurfactants prepared by attaching a dipeptide D-alanyl-D-alanine or a racemic dipeptide DL-alanyl-DL-alanine to cholesteryl-chloroformate was employed for affinity reverse micellar extraction of vancomycin. The forward extraction efficiency increases significantly in the presence of an affinity cosurfactant. The recovery of vancomycin from fermentation broth with high selectivity was also achieved by employing this affinity cosurfactant.