The bisecting N-acetylglucosamine (GlcNAc) structure, characterized by β1, 4-linked GlcNAc attached to the core β-mannose residue, represents a distinctive form of N-glycosylation modification. Bisecting GlcNAc modification is implicated in numerous biological processes such as cell adhesion, fertilization and embryonic development, neurogenesis, immune responses, and tumorigenesis. This review elucidates the formation mechanisms of bisecting GlcNAc structure and highlights its diverse biological functions in the nervous system, immune response, tumor metastasis and development. Furthermore, it provides an overview of the technological advances in analyzing bisecting GlcNAc structure, providing scientific references for corresponding biological research and drug development.