Ulcerative colitis (UC) is a recurrent inflammation of the gastrointestinal tract, for which available treatment drugs are severely limited. Natural polysaccharides show potential for UC prevention. Herein, we extracted TCP80-1, a polysaccharide with significant anti-ulcerative colitis (UC) activity, from Tupistra chinensis Baker for the first time. Structure analysis revealed that TCP80-1 (3190 Da) was a levan-neoseries fructan containing → 6)-α-D-Glcp-(1→, →1)-β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, →6)-β-D-Fruf-(2→ and β-D-Fruf-(2→ residues as backbone, with →6)-β-D-Fruf-(2→, and β-D-Fruf-(2→ as side chains substituted at C-6. The protective effect of TCP80-1 on UC was further evaluated using a UC Drosophila melanogaster model. The results demonstrated that TCP80-1 could alleviate the UC symptoms by reducing colonic atrophy, enhancing intestinal barrier, and promoting the proliferation and differentiation of intestinal stem cells (ISCs) into intestinal epithelial cells (IECs). Our findings provide important structural information about TCP80-1 and establish the foundation for the future progression and utilization of T. chinensis polysaccharide within the realm of UC therapy.