Abstract: Human milk oligosaccharides (HMOs) are abundant components in human milk, playing a pivotal role in shaping the characteristic gut microbiota and regulating immune function in infants and other populations. This review elaborates on the structure, content, detection methods, biological functions, and food applications of HMOs, while contrasting the differences between animal milk oligosaccharides and human milk oligosaccharides. Furthermore, it explores compounding strategies for infant formula and analyzes regulatory advancements in this field. By synthesizing these insights, this review aims to provide researchers and developers in the food industry with a practical, forward-looking, and instructive reference to advance the application of HMOs in the food sector.