An ultrasonic‐assisted liquid–liquid extraction based on the aqueous two‐phase system (ATPS) was applied for the first time in this study for the extraction of flavonoids from Tibetan sea‐buckthorn (Hippophae thibetana S) fruit. The response surface method provided the optimum values of several experimental variables. The best extraction yield of 8.30 ± 0.03% was obtained for two cycles of extraction, using an ultrasound bath temperature of 50°C and liquid–solid ratio 99:1. The antioxidant capacity of the extract was assessed by the in vitro DPPH and ABTS assays, with the IC50 values similar to or better than ascorbic acid (0.343 mg/ml and 0.164 mg/ml, respectively). This study revealed the great potential of ultrasound‐aided ATPS as the green alternative for the extraction of flavonoids from H. thibetana S. The high antioxidant activity of the extract indicates its applicability as a natural functional ingredient for food and other industries. Practical applications In this study, ethanol and ammonium sulfate were selected as extraction and separation solvents to optimize the extraction process of flavonoids from sea buckthorn fruits, which is simpler and more environmentally friendly than the traditional method, while reducing the process cost, and has great application value. In addition, the analysis of the extracted components of edible sea buckthorn fruits revealed that the flavonoids contained in sea buckthorn fruits have antioxidant activity close to that of vitamin C. Combined with the above studies, sea buckthorn fruits have the potential to be developed into functional foods and additives with antioxidant functions.