The ultrasound-assisted extraction process of polysaccharides from the bee pollen of wolfberry was optimized by response surface methodology based on Box-Behnken design with the extraction rate as the observation indexes. The antioxidant activity of polysaccharides extracted by the optimal process was studied in vitro. The optimal extraction conditions were obtained as follows:solid-liquid ratio 1∶25 (g/mL), temperature 90℃, ultrasonic power 240W, and ultrasonic time 20min. Under these conditions, the yield of polysaccharides was 0.89%-0.91%, which was insistent with the predicted value, and it indicated that the model was good and the optimization process was feasible. The results of antioxidant activity in vitro were showed that the polysaccharide extracted from bee pollen of wolfberry had good DPPH radicals and ABTS⁺· scavenging activities, however, the FRAP value was low. The results provided a basis for the research, development, and utilization of polysaccharide extracted from bee pollen of wolfberry.