Seedless chestnut rose (Rosa sterilis S. D. Shi, RS) is a fresh type of R. roxburghii Tratt with copious functional components in its fruit. Polysaccharides are recognized as one of the vital bioactive compounds in RS fruits, but their antioxidant and hypoglycemic properties have not been extensively explored. Hence, in this study, accelerated solvent extraction (RSP-W), citric acid (RSP-C), 5% sodium hydroxide/0.05% sodium borohydride (RSP-A), and 0.9% sodium chloride (RSP-S) solution extraction were individually utilized to obtain RS fruit polysaccharides. The physicochemical properties, structural characteristics, and biological activities were then compared. Results indicated that extraction methods had significant influences on the extraction yield, uronic acid content, monosaccharide composition, molecular weight, particle size, thermal stability, triple-helical structure, and surface morphology of RSPs apart from the major linkage bands and crystalline characteristics. The bioactivity tests showed that the RSP-S, which had the greatest amount of uronic acid and a comparatively lower molecular weight, exhibited more potent antioxidant and α-glucosidase inhibitory property. Furthermore, all RSPs inhibited α-glucosidase through a mixed-type manner and quenched their fluorescence predominantly via a static quenching mechanism, with RSP-S showing the highest binding efficiency. Our findings provide a theoretical basis for utilizing RSPs as functional ingredients in food industries.