Polysaccharides are biopolymers known to possess various bioactivities. Because of their molecular complexity, the structural characterization of polysaccharides remains challenging, and difficult to be completed with a single analytical method. In this study, a novel approach for the characterization of linkages and anomeric configuration of polysaccharides was proposed. Based on ion mobility-mass spectrometry (IM-MS), a database containing 5 glucotriose standards was set up. Information about the arrival time distribution and fragmentation patterns of these standards were included. The method was validated by three commercially available purified polysaccharides, namely laminarin, dextrin, and dextran, each having distinct connectivity and configuration of the glycosidic bonds. Lastly, the method was successfully applied to analyze polysaccharides prepared from three medicinal mushrooms, namely Xylaria nigripes, Grifola frondosa, and Laetiporus sulphureus. The results showed that water-soluble non-digestible polysaccharides of X. nigripes and G. frondosa were mainly composed of (1→3)-β-glucan, while that of L. sulphureus was composed of (1→3)-ɑ-glucan. The present method has the advantages of being simple in sample preparation and short analysis time.