Abstract: In order to obtain the optimal conditions of ultrasound-assisted aqueous two-phase extraction of polyphenols from mango seed kernel, a novel extraction method involving ultrasound-assisted extraction combined with propyl-alcohol ammonium sulfate aqueous two-phase separation was developed for the extraction of polyphenols from mango seed kernel. Response surface methodology, based on a five levels, four variables central composite designs was employed to study the effect of extraction conditions on the extraction yield of total polyphenols. An extraction yield of 6.67% was achieved under the optimal extraction conditions of 59.7% of propyl-alcohol volume fraction, 23.4% of ammonium sulfate investigated concentration, 27.0 min of extraction time and liquid/solid ratio of 23 mL/g at ultrasonic power of 400 W and temperature of 45℃, which was agreement well with the optimal prediction value (6.82%). Total polyphenols content in the extract of ultrasound-assisted aqueous two-phase extraction was higher than that of conventional ultrasound-assisted extraction. The research indicates that the method of combining aqueous two-phase separation with ultrasound-assisted extraction to improve the extraction of polyphenols is practicable. The research can provide a reference for full utilization of mango seed kernel and the extraction technique of polyphenols.