Polysaccharides from Dendrobium officinale have garnered attention for their diverse and well-documented biological activities. In this study, we isolated three ethanol-fractionated polysaccharides from Dendrobium officinale (EPDO) and investigated their digestive properties and effects on gut microbiota regulation in vitro. The results indicated that after simulating digestion in saliva, gastric, and small intestinal fluids, three EPDOs, EPDO-40, EPDO-60 and EPDO-80, with molecular weights (Mw) of 442.6, 268.3 and 50.8 kDa, respectively, could reach the large intestine with a retention rate exceeding 95%. During in vitro fermentation, the EPDOs were broken down in a “melting” manner, resulting in a decrease in their Mw. EPDO-60 degraded more rapidly than EPDO-40, likely due to its moderate Mw. After 24 h, the total production of short-chain fatty acids (SCFAs) for EPDO-60 reached 51.2 ± 1.9 mmol/L, which was higher than that of EPDO-80. Additionally, there was an increase in the relative abundance of Bacteroides, which are capable of metabolizing polysaccharides. EPDO-60 also promoted the growth of specific microbiota, including Prevotella 9 and Parabacteroides, which could potentially benefit from these polysaccharides. Most notably, by comparing the gut microbiota produced by different fermentation carbon sources, we identified the eight most differential gut microbiota specialized in polysaccharide metabolism at the genus level. Functional prediction of these eight differential genera suggested roles in controlling replication and repair, regulating metabolism, and managing genetic information transmission. This provides a new reference for elucidating the specific mechanisms by which EPDOs influence the human body. These findings offer new evidence to explain how EPDOs differ in their digestive properties and contribute to the establishment of a healthy gut microbiota environment in the human body.