This study presents an efficient method for the extraction of polysaccharides from Ulva lactuca using ultrasound-assisted multi-enzyme extraction. The optimal enzyme mixture was determined as cellulase (35.3%), pectinase (34.5%), and alkaline protease (30.2%), which increased the polysaccharide yield from 6.43% to 26.68%. Further optimization using response surface methodology (RSM) identified the optimal conditions: enzyme concentration of 1.5%, enzymatic time of 1.1 h, ultrasonic time of 90 min, and enzymatic temperature of 60°C, resulting in a polysaccharide yield of 30.14%. The extracted polysaccharides exhibited strong antioxidant activity in various models, including DPPH, ABTS, hydroxyl, and superoxide radical scavenging, as well as protection against H₂O₂-induced cellular damage. The study provides a solid foundation for the efficient extraction and application of Ulva polysaccharides in biomedicine and food. The method is environmentally friendly, cost-effective, and improves the extraction yield and biological activity of Ulva polysaccharides.This study presents an efficient method for the extraction of polysaccharides from Ulva lactuca using ultrasound-assisted multi-enzyme extraction. The optimal enzyme mixture was determined as cellulase (35.3%), pectinase (34.5%), and alkaline protease (30.2%), which increased the polysaccharide yield from 6.43% to 26.68%. Further optimization using response surface methodology (RSM) identified the optimal conditions: enzyme concentration of 1.5%, enzymatic time of 1.1 h, ultrasonic time of 90 min, and enzymatic temperature of 60°C, resulting in a polysaccharide yield of 30.14%. The extracted polysaccharides exhibited strong antioxidant activity in various models, including DPPH, ABTS, hydroxyl, and superoxide radical scavenging, as well as protection against H₂O₂-induced cellular damage. The study provides a solid foundation for the efficient extraction and application of Ulva polysaccharides in biomedicine and food. The method is environmentally friendly, cost-effective, and improves the extraction yield and biological activity of Ulva polysaccharides.