High-performance separation and purification present essential obstacles for the application of biosurfactant mannosylerythritol lipids (MEL) in many fields. In this study, we developed an extraction process, which efficiently isolates MEL from fermentation broth with the solvent system methanol/water/n-hexane. At pH 2, extraction with methanol/water/n-hexane at a ratio of 2:1:1 recovers about 80% MEL, and the following extraction with methanol/water/n-hexane at ratio of 3:1:1 can further recover about 14% MEL. After that, MEL were purified using a methanol/n-hexane mixture with a ratio of 1:1. The whole presented extraction process recovers over 90% MEL with high purity, excellent surface activities, excellent surface activity (reduce the surface tension of water to below 28 mN/m at a concentration of 15 mg/L), and high emulsification activity (E-24 of 85-96%). This efficient and easy-to-operate separation process has the potential to contribute to industrial applications of MEL in many fields requiring high purity.