To establish the quality control method of Dioscorea opposita Thunb., the multi-level fingerprinting of polysaccharides was established and the relationship between fingerprint and immune activity was analyzed. The two molecular weight segments Mw1 (1.38 × 105-1.63 × 106 Da) and Mw2 (3.27 × 103-4.37 × 103 Da), thirteen infrared absorption peaks (3399.26 cm-1, 2929.32 cm-1, 1631.78 cm-1, 1400.39 cm-1, 1351.80 cm-1, 1123.58 cm-1, 1024.76 cm-1, 931.53 cm-1, 854.76 cm-1, 760.43 cm-1, 708.14 cm-1, 616.47 cm-1, and 526.78 cm-1), and four monosaccharides (Man, Rha, GalA, and Glc) were used to evaluate the quality of Dioscorea opposita Thunb. The molecular weight fragments of Mw1, FT-IR absorption peaks of 1631.78 cm-1, and two monosaccharides (Man and Glc) would be used to identify Dioscorea opposita Thunb. polysaccharide (DOP) from different origins. The relationship of spectrum-effect showed that polysaccharides with features such as higher Mw1, a lower peak height of 1631.78 cm-1, higher content of Man, and lower content of Glc exerted stronger immune activity. In conclusion, this study established a polysaccharide-based quality evaluation method for Dioscorea opposita Thunb. and explored the relationship between polysaccharide fingerprints and in vitro immune activity, which provided a basis for further research on Dioscorea opposita Thunb.