Abstract: In this study, the ultrasound-assisted extraction conditions of total flavonoids were optimized, and the structure and composition of total flavonoids were further analyzed. On the basis of single factor experiments, orthogonal experiments was carried out on four factors, including solid-liquid ratio, extraction time, temperature and power to obtain the optimal extraction conditions based on extraction yield. Under these conditions, total flavonoids were purified by D101 macroporous resin. The structure of total flavonoids before and after purification was characterized by UV-Vis and Fourier transform infrared spectroscopy (FT-IR). Finally, the constituents of the purified compounds were analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The results showed that the optimal extraction conditions were as follows: Ethanol concentration of 60%, solid-liquid ratio of 1:25 mg/mL, ultrasonic temperature of 45 ℃, time of 120 min, power of 250 W, and extraction yield of 2.776%. The characteristic structure of total flavonoids was analyzed by UV-Vis spectroscopy (UV-Vis). Fourier transform infrared spectroscopy (FT-IR) also showed that the total flavonoids contained O-H, C-H, C=O, C=C, phenolic hydroxyl groups and other characteristic functional groups, which was consistent with the typical structure of flavonoids. Thirty flavonoids were detected in the purified products by UPLC-MS/MS, including citrinin, hesperetin, azalea, protocatechualdehyde, diosmin, naringin chalcone, sweet orange flavone, naringin and rutin. The relative content of citrinin was the highest, which was 50.854%±0.089% . This study would provide a theoretical basis for the functional development and utilization of crystal Mesembryanthemum crystallinum.