Polysaccharides are promising biomolecules with lowtoxicity and diverse bioactivities in food processing and clinical drug development. However, an essential prerequisite for their applications is the fine structure characterization. Due to the complexity of polysaccharide structure, partial degradation is a powerful tool for fine structure analysis, which can effectively provide valid information on the structure of backbone and branching glycosidic fragments of complex polysaccharides. This review aims to conclude current methods of partial degradation employed for polysaccharide structural characterization, discuss the molecular mechanisms, and describe the molecular structure and solution properties of degraded polysaccharides. In addition, the effects of polysaccharide degradation on the conformational relationships between the molecular structure and bioactivities, such as antioxidant, antitumor, and immunomodulatory activities, are also discussed. Finally, we summarize the prospects and current challenges for the partial degradation of polysaccharides. This review will be of great value for the scientific elucidation of polysaccharide fine structures and potential applications.