Dendrobium officinale oligosaccharides (DOO), as a novel prebiotic, offer promising therapeutic potential for the treatment of inflammatory diseases. In this study, the alleviating effect of DOO on dextran sulfate sodium (DSS)-induced chronic colitis in mice was investigated. DOO treatment relieved the main symptoms of weight loss, colon shortening, and bloody stool caused by colitis. DOO ameliorated histopathological colon tissue damage and reduced levels of pro-inflammatory factors (TNF-α, IL-6, IL-1β). Western blot results suggested that DOO downregulated the phosphorylation levels of key proteins in the NF-κB signaling pathway, including IKKα/β, IκBα and p65. 16S rRNA sequencing suggested that DOO altered the diversity and composition of gut microbiota in DSS-induced mice. It promoted the intestinal colonization of beneficial bacteria such as Dubosiella, Lactobacillus and Alistipes, and inhibited the abnormal overgrowth of Akkermansia. Furthermore, DOO increased the secretion of short-chain fatty acids (SCFAs) such as acetic acid, propionic acid, and butyric acid to maintain intestinal homeostasis. These findings suggest the potential of DOO as a therapeutic agent for ulcerative colitis (UC).