Oligosaccharides from seaweed have recently attracted attention for their prebiotic potential in foods. In this study, ulvan polysaccharides (UPS) obtained by hot water extraction were treated with ulvan lyase 24A and hydrolyzed to ulvan oligosaccharide (UOS). The composition of UOS was analyzed for total sugars, sulfate, uronic acid, protein, reducing sugars, monosaccharide composition, and molecular weight distribution. The FT-IR spectrum showed that the enzyme did not remove the specific functional groups when UPS was degraded to UOS. The prebiotic effect of UOS was investigated by its influence on the growth of three lactic acid bacteria. UOS promoted the growth of Lactobacillus acidophilus and Lactobacillus plantarum better than commercial prebiotic galacto-oligosaccharide (GOS) and suppressed the growth of pathogens (Bacillus cereus and Escherichia coli). The protective effect of UOS under stimulated gastrointestinal conditions was also studied. It was found that L. plantarum grown in a medium containing UOS showed the highest survival rate in the presence of α-amylase, while L. acidophilus showed the highest survival rate under HCl conditions. In addition, all strains of LAB, grown in medium supplemented with UOS, showed the lowest survival rates. The present study demonstrated that UOS obtained from U. rigida can be used as a prebiotic additive that has significant potential for the food and feed industry.