Agaricus bisporus (A. bisporus) is a rich source of polysaccharides with medical and nutraceutical interests; however, the complex structure of A. bisporus polysaccharides (ABPs) poses a challenge to its quality control. This study aims to investigate the immunomodulatory fractions of ABPs and further establish a quality control system for the detection and characterization of bioactive ABPs. Two fractions of ABPs were obtained using gradient alcohol precipitation combined ultrafiltration, followed by characterization of molecular weight, monosaccharide composition, chemical structure, and evaluation of immunomodulatory activity. Results showed that the two isolated fractions with different molecular weight, 8.95 × 10 Da (ABP17-1) and 1.73 × 10 Da (ABP17-2), were more homogeneous and exerted stronger immunomodulatory activity than that of ABPs. Furthermore, an integrated fingerprint technique including HPSEC-MALL-RID and HPLC was applied for the fingerprint analysis of ABPs from 17 batches of A. bisporus, which showed high similarity. Besides, two polysaccharide fractions, two monosaccharides (glucose and galactose), and the main cellulose hydrolysate glucose were identified as potential quality control markers of ABPs. Overall, this study could help better understand the structure–activity relationship of ABPs and serve as a guidance for the quality control of active ABPs and their related products.