Glycans in human milk serve several important biological functions that promote infant health. As kind of important glycans, glycosaminoglycans (GAGs) are a complex family of polyanionic carbohydrate, participating in a variety of critical physiological and pathological processes. In this study, the content and the detailed composition of human milk GAGs from Chinese mothers in different stages of lactation, based on a liquid chromatography-tandem mass spectrometry approach was investigated. The results showed that the GAG fraction in the human milk samples was very complex as it was composed of heparan sulfate, chondroitin sulfate, and hyaluronic acid. With lactation extending, the total amount of GAGs in human milk decreased. This study provided an important guide for the demands of GAGs during different stages of lactation. The results were also beneficial for studies on the composition and functional properties of infant formula.