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The ultrasonic-assisted aqueous two-phase extraction (UAATPE) of flavonoid glycosides from Malvaviscus arboreous Cav. flower (MACF) was developed using ethanol/ammonia sulfate systems, followed by the ultrasonic-assisted acid hydrolysis (UAAH) of the top extract with HCl solution. The optimization of UAATPE and UAAH processes was accomplished by single-factor experiments and response surface methodology. As a result, the flavonoid glycosides enriched in the top phase could achieve a maximum yield of 35.9 ± 1.1 mg/g by UAATPE and were completely hydrolyzed by UAAH deglycosylation. The flavonoid glycosides and their hydrolyzates were separated and characterized by high-performance liquid chromatography and ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Ultrasonic enhancement of the extraction and hydrolysis was explored by comparative study. Furthermore, the in vitro activity of the flavonoid glycosides and the aglycones were comprehensively evaluated by antioxidant activity assays, including ferric-reducing antioxidant power and scavenging DPPH, hydroxyl, and superoxide radicals. All of the IC50 values suggest that the antioxidant activity of flavonoid aglycones was stronger than that of their glucosides and even vitamin C, revealing that the deglycosylated flavonoids from MACF were the more powerful antioxidants. This study provided an effective and eco-friendly strategy for the extraction, separation, and purification of flavonoids from MACF, as well as for the development of the potential flavonoid antioxidants.