Yan Meng, Mengying Yao, Xiaojuan Xu
Polysaccharides are the third most diverse biopolymers in nature, after proteins or nucleic acids. Structure determines activity. Increasing evidence has shown that the conformations of polysaccharides strongly influence their physicochemical properties and bioactivities. Elucidating the stiffness, flexibility, and detailed chain parameters can lead researchers to better apply polysaccharides in the fields of food, pharmaceuticals, and materials science. Nowadays, light scattering methods (LLS/DLS/SEC-MALS), microscopy technologies (AFM/TEM/SEM), and computational modeling (MD) have provided valuable insights into chain dimensions, conformation transformation, and aggregation behaviors in solution. This review summarizes the major analytical techniques used to investigate polysaccharide conformation, including spectroscopic, scattering, microscopic, hydrodynamic, and computational approaches. We hope to provide a practical guide for researchers to explore polysaccharide conformations, establish the structure–activity relationship, and advance the applications of polysaccharides in the food, cosmetic, and biomedical fields.