Abstract: The optimal extraction process of phytoglycogen (PG) from sweet corn by ultrasound-assisted method was studied, and the physicochemical properties and biological activities of PG were evaluated in this paper. The effects of solid-liquid ratio, ultrasonic power and ultrasonic time on the yield of PG were investigated and the process parameters were further optimized by orthogonal experiment. Furthermore, the physicochemical properties of PG obtained by ultrasound-assisted method were investigated. Meanwhile, the antioxidation, cytotoxicity, apoptosis and hemolysis of PG were evaluated in vitro. The results showed that the optimal extraction conditions were as follows: Solid-liquid ratio of 1:9 (g/mL), ultrasonic power of 160 W and ultrasonic time of 80 min, the actual yield was 14.31%±0.38%. The PG particle size obtained by ultrasound-assisted method was lower than that obtained by traditional water extraction and alcohol precipitation method. In vitro antioxidant experiment showed that PG had a certain antioxidant capacity, especially it was more sensitive to DPPH free radicals, and the clearance rate reached 54.58%±1.39% at 20 mg/mL. The results of biosafety evaluation showed that the survival rate of mouse fibroblasts (3T3) was higher than 95% after incubation with different concentrations of PG for 24 or 48 h. Meanwhile, PG had no obvious apoptotic effect on cells, and the apoptotic rate was lower than 7% at the maximum concentration. After 2 h of co-incubation, the hemolysis rate caused by PG on red blood cells was also much lower than the national standard (5%), which confirmed that PG had good biocompatibility with normal cells and red blood cells. Overall, this study showed that ultrasound-assisted method could improve the yield and physicochemical properties of PG, and also confirmed that PG was a non-toxic natural nanoparticle, which had great application potential in drug or food factor delivery.