Milk oligosaccharides have long received great attention to their high potencies of anti-infective, anti-inflammatory, and prebiotic activities. As a result, the structures of more than 130 human milk oligosaccharides have been reported until now. In order to investigate the structure, function, and application of milk oligosaccharides, the pure oligosaccharides are needed in large quantities. In this article, the syntheses by the transglycosylation reactions using glycosidase and the syntheses of higher oligosaccharides by glycosyltransferase are described in detail. In addition, the example of the syntheses by the genetically engineered metabolic systems are also demonstrated. Furthermore, the isolation of milk oligosaccharides from mammalian milk by the improved process of the traditional charcoal column method are described.