The physicochemical properties and prebiotic potential of polysaccharide extract from the green seaweed Ulva Rigida were explored. The UPS PII-1 fraction purified by DEAE-Sephacel and Sephacryl S-400 columns was identified as a high molecular weight heteropolysaccharide (1.63 × 103 kDa) composed of rhamnose, glucose, mannose, galactose, arabinose, and fructose. FTIR analysis showed typical absorption bands of the structure of ulvan polysaccharides. In vitro prebiotic studies revealed that U. rigida polysaccharides (UPS) were highly resistant to gastric and intestinal juice hydrolysis. UPS also enhanced the growth of probiotic bacteria including Bifidobacterium and Lactobacillus as a potential source of prebiotics in functional food.