Human milk oligosaccharides are one of the most critical components of breast milk， with valuable functions such as regulating immunity and promoting brain development， and they have significant commercial potential in infant nutrition and medical therapy. The rapidly growing market demand has put new requirements on their supply， and new production methods of human milk oligosaccharides have emerged. The biosynthetic routes have broken traditional barriers and brought new opportunities and challenges to the industry. This review first introduced the basic properties and industrial application of human milk oligosaccharides， then focused on the research advances in the biosynthesis pathway of human milk oligosaccharides and discussed in depth the key technologies， advantages， and pain points of the chemoenzymatic method and microbial cell factory in their synthesis. Finally， the future outlook of human milk oligosaccharides had been prospected.