Human milk oligosaccharides (HMOs) present in breast milk are a family of highly complex oligosaccharides that play a positive role in the intestinal flora, immune barrier, and brain development of infants. Due to the complex matrix in breast milk, the variety of oligosaccharides, the span of abundance, and the presence of many isomers, the detection of breast milk is faced with many challenges. A variety of techniques have been used for the analysis of human milk oligosaccharides and over 200 HMOs have been identified. Liquid chromatography and capillary electrophoresis have been effective in separating human milk oligosaccharides. Nuclear magnetic resonance spectroscopy, mass spectrometry and infrared multiphoton dissociation spectroscopy have promoted the comprehensive structural resolution of HMOs. This paper reviews the various techniques used to achieve high sensitivity and specificity for the analysis of HMOs and compares the advantages and disadvantages of the different techniques. It also focuses on the breakthroughs in mass spectrometry and the combination of different techniques to facilitate the analysis and determination of HMOs, providing a comprehensive technical support to explore the structure-function relationship of oligosaccharides and to gain a deeper understanding of the biological functions of HMOs.