A Box-Behnken design (BBD) of response surface methodology (RSM) is employed to further optimize ultrasonic-assisted extraction conditions for Ophiopogon japonicus polysaccharides. The results show that optimized extraction conditions are liquid-solid ratio of 22:1 mL g(-1), ultrasonic time of 27 min, and ultrasonic power of 200 W. Under these conditions, the mean experimental value of extraction yield (39.49%) is achieved, which is 1.33 times that of distilled water leaching extraction (29.68%). In addition, one of the eluting fractions, SPs-1, has high alpha-glucosidase and alpha-amylase inhibitory activities as well as 1,1-Diphenyl-2-picrylhdrazyl radical and alpha-glucosidase, 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) cation scavenging activities with an IC50 value of 0.060, 0.021, 0.50, and 0.10 mg mL(-1). Therefore, SPs-1 from O. japonicus can be considered as multifunctional biological ingredients to be used in anti-hyperglycemic pharmaceutical formulation and antioxidant in functional foods.