Human milk oligosaccharides (HMOs) are complex glycans that are the third largest solid component in human milk. It has attracted great interest in recent years due to their critical role in boosting infant health. These oligosaccharides play an important role in a variety of physiological processes, such as shaping the infant gut microbiome, preventing pathogenic infections and promoting the development of immune system. However, limited availability of HMOs hampered their use in food and medical areas. Moreover, most of the HMOs are unique to human milk and difficult to isolate. The strategies, chemical synthesis, whole-cell fermentation, and purification from human milk, have their advantages and come with their own challenges. In this review, we examined the remarkable progress that has been made in the enzymatic and chemoenzymatic synthesis of HMOs, and discussed the challenges and opportunities in large-scale synthesis of HMOs.