The partitioning behaviors of vancomycin in a temperature-induced nonionic surfactant two-phase system were studied. N-Decyltetra(ethylene oxide) (C10E4) was employed as the nonionic surfactant. Vancomycin had a preference for the micelle-rich top phase under most experimental conditions. However, at pH 4 vancomycin preferred to stay in the micelle-poor bottom phase. An affinity cosurfactant, D-alanyl-D-alanine modified cholesterol, was employed in the extraction system to increase the partition coefficient of vancomycin. The I,partition coefficient increased significantly from 0.87 to 15.98 when an affinity cosurfactant was employed. Vancomycin could form a micelle, and its critical micelle concentration (CMC) was determined by the methyl orange binding technique and the surface tension method. The micelle formation of vancomycin was considered to induce the lower partition coefficient and a significant increase of the partition coefficient in the presence of the affinity cosurfactant at pH 4. Direct recovery of vancomycin From fermentation broth using affinity extraction was also demonstrated.