Polysaccharides from alfalfa (Medicago sativa L., MSPs) were extracted by four different methods: hot-water extraction (HWE), ultrasonic-assisted extraction (UAE), enzyme-assisted extraction (EAE) and ultrasonic-enzyme-assisted extraction (UEAE). The experimental results showed that the monosaccharide compositions of the four MSPs extracted by the different extraction methods were the same, but the monosaccharide proportions were different. MSPs extracted by the UEAE method possessed the largest extraction yield, the highest uronic acid content and the best antioxidant activities among the four MSPs analysed. Additionally, the Plackett-Burman design (PBD) and response surface methodology (RSM) were used to determine the optimum UEAE conditions for extracting MSPs, revealing optimal conditions at a pH of 5.7, a 58 min enzymolysis time, a 40 degrees C enzymolysis temperature and a 41 degrees C ultrasonic temperature; an MSPs yield of 12.95% was obtained under these extraction conditions.