Quality analysis of edible mushrooms based on polysaccharides is generally difficult due to their complicated structures and hard separation. Here, multiple fingerprint analysis of polysaccharides based on chromatographic and spectrometric techniques were developed, and then applied in comparative analysis of Auricularia heimuer (AH), Auricularia cornea (AC), Auricularia cornea 'Yu Muer' (ACY) and Tremella fuciformis (TF). Firstly, polysaccharides were obtained with the molecular weights between 1.783 × 10 and 6.774 × 10 Da. Then, complete hydrolysis by TFA and enzyme digestion by cellulase were employed and subsequently analyzed by HPLC-UV, GC-MS, HILIC-HPLC-ELSD and HILIC-HPLC-ESI-HCD-MS/MS, and ATR-FT-IR were used to characterize the functional groups of intact polysaccharides. By chemometric analysis, differential markers of d-xyl, l-fuc, l-arb, d-glc, disaccharide and hexasaccharide were selected, and AC and ACY were proved to be same species from the viewpoint of polysaccharides firstly. Furthermore, the structures of oligomers with DPs of 2-8 and →4)-β-d-Glcp-(1→ unit with different contents were inferred by combinatory analysis of ESI-MS/MS, glycosidic linkage, monosaccharide compositions and functional groups. In conclusion, the combinatory method of multiple fingerprint and pattern recognition is powerful not only for structural elucidation of polysaccharides, but also for quality analysis and species differentiation of edible mushrooms from the perspective of biological polysaccharides.