Recently, Dendrobium officinale polysaccharide (DOP), a typical acetylated glucomannan, has been widely applied in functional foods owing to its excellent bioactivity. However, the insufficiency of studies on in vivo process severely limits the further utilization of DOP. The aim of this study was to systematically investigate the gastrointestinal digestive behaviour of DOP after oral administration by labelling it with two fluorescein aminopyrene-1,3,6-trisulfonic acids, trisodium salt (APTS) and cyanine 7.5 (Cy7.5). Combining the results of NIR imaging and HPGPC, we found that DOP was poorly absorbed directly in the prototype form; instead, DOP moved with the intestinal contents to the distal part of the intestine, where Bacteroides aggregated for a prolonged time and was metabolized to oligosaccharide-like substances. In contrast, the digestive degradation of DOP in pseudo-sterile mice with a targeted clearance of Bacteroides significantly weakened, which provided the basis and direction for the subsequent search for more specific metabolic pathways of DOP in vivo.