Ulcerative colitis (UC) is a global intestinal disease, and conventional therapeutic drugs often fail to meet the needs of patients. There is an urgent need to find efficient and affordable novel biological therapies. Saccharomyces boulardii has been widely used in food and pharmaceutical research due to its anti-inflammatory properties and gut health benefits. However, there is still a relatively limited comparison and evaluation of different forms of S. boulardii treatment for UC. This study aimed to compare the therapeutic effects of S. boulardii, heat-killed S. boulardii, and S. boulardii β-glucan on UC, to explore the potential of heat-killed S. boulardii as a new biological therapy. The results demonstrate that all three treatments were able to restore body weight, reduce the disease activity index (DAI), inhibit splenomegaly, shorten colon length, and alleviate histopathological damage to colonic epithelial tissues in DSS-induced colitis mice. The oral administration of S. boulardii, heat-killed S. boulardii, and S. boulardii β-glucan also increased the levels of tight junction proteins (Occludin and ZO-1), decreased the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the serum, and suppressed the expressions of TNF-α, IL-1β, and IL-6 mRNA in the colon. In particular, in terms of gut microbiota, S. boulardii, heat-killed S. boulardii, and S. boulardii β-glucan exhibited varying degrees of modulation on DSS-induced dysbiosis. Among them, heat-killed S. boulardii maximally restored the composition, structure, and functionality of the intestinal microbiota to normal levels. In conclusion, heat-killed S. boulardii showed greater advantages over S. boulardii and S. boulardii β-glucan in the treatment of intestinal diseases, and it holds promise as an effective novel biological therapy for UC. This study is of great importance in improving the quality of life for UC patients and reducing the burden of the disease.