Despite the well-known health benefits of unsaturated alginate oligosaccharides (UAOS), limited information exists on how they regulate the gut microbiota and intestinal mucosal immunity. In this study, UAOS was produced by alginate lyase degradation. Fourier transform infrared (FTIR), mass spectrometry (MS), and nuclear magnetic resonance (NMR) analyses showed that UAOS primarily consists of oligosaccharides, mainly pentamers, with a G/M ratio of 1.44 and unsaturated double bonds at the non-reducing end. UAOS exhibited good prebiotic effects; increased beneficial intestinal bacteria; improved the diversity, structure, and composition of the gut microbiota; and promoted the production of SCFAs. In particular, UAOS significantly increased the abundance of butyrate levels and their producing microbiota, such as Lachnospiraceae, Alloprevotella, and Butyicicoccus. Moreover, orally administered UAOS alleviated intestinal mucosal immunosuppression by upregulating the levels of the tight junction proteins occludin and ZO-1, enhancing the intestinal biochemical and immune barrier function by increasing levels of mucin-2 and SIgA, upregulating the CD4+/CD8+ ratio, regulating CD4+ T cell differentiation, and stimulated immune cytokine secretion and transcription factor production (T-bet/GATA-3). This process was related to TLR4/MyD88/NF-κB pathway. In summary, UAOS effectively regulates intestinal mucosal immune homeostasis by strengthening the intestinal barrier and regulating the intestinal microbiota and intestinal butyrate levels. Therefore, UAOS acts as a prebiotic and immune stimulator to improve host health.