Artemisia argyi leaf polysaccharides (AALPs) were extracted using ultrasound-assisted extraction (UAE) and evaluated for antifatigue activity. Response surface methodology (RSM) optimized UAE conditions: 300 W ultrasonic power, 51°C temperature, 20 mL/g liquid:solid ratio, and 47 minutes of ultrasonication, yielding a maximum extraction rate of 10.49%. UAE produced higher yields and total sugar, uronic acid, and sulfate contents compared to hot water extraction (HWE). AALP-U, obtained via UAE, showed greater stability, antioxidant activity, and antifatigue potential than AALP-H from HWE. In mouse studies, AALP-U significantly prolonged swimming time, increased liver and muscle glycogen levels, and improved biochemical indices, indicating its potential as a functional food ingredient. AALP-U also reduced oxidative stress markers like ROS, SOD, and MDA, suggesting its antifatigue mechanism involves antioxidant activity. The study highlights UAE's efficiency in extracting AALPs with superior properties, offering a sustainable method for polysaccharide extraction and application in food and medicine.Artemisia argyi leaf polysaccharides (AALPs) were extracted using ultrasound-assisted extraction (UAE) and evaluated for antifatigue activity. Response surface methodology (RSM) optimized UAE conditions: 300 W ultrasonic power, 51°C temperature, 20 mL/g liquid:solid ratio, and 47 minutes of ultrasonication, yielding a maximum extraction rate of 10.49%. UAE produced higher yields and total sugar, uronic acid, and sulfate contents compared to hot water extraction (HWE). AALP-U, obtained via UAE, showed greater stability, antioxidant activity, and antifatigue potential than AALP-H from HWE. In mouse studies, AALP-U significantly prolonged swimming time, increased liver and muscle glycogen levels, and improved biochemical indices, indicating its potential as a functional food ingredient. AALP-U also reduced oxidative stress markers like ROS, SOD, and MDA, suggesting its antifatigue mechanism involves antioxidant activity. The study highlights UAE's efficiency in extracting AALPs with superior properties, offering a sustainable method for polysaccharide extraction and application in food and medicine.