The discovery of a green extraction solvent for natural plants could promote related research. In this study, deep eutectic solvents (DES) were used as green solvents coupled with an ultrasound-assisted extraction method (UAE) to extract flavonoids from lotus leaves. Thirty-four different DES were performed and choline chloride/urea with 40% water was chosen as the most promising one, and the related parameters in the procedures were optimized, resulting in the highest extraction amount of flavonoids in lotus leaves. D-101 was selected from four macroporous resins to separate the flavonoids from DES. Moreover, DES could be recycled and efficiently reused four times with satisfactory performances. In addition, the lotus leaf flavonoids from the DES extract exhibited antioxidant activities in five kinds of assays including DPPH, ABTS, Fe3+ reducing, FRAP, and Fe2+ chelating. It also showed antibacterial activities on Staphylococcus aureus and Escherichia coli bacterial strains with minimal inhibitory concentrations at 1666 μg/mL and 208 μg/mL, respectively. In the HPLC analysis, the three main components in the DES extract were identified as astragalin, hyperoside, and isoquercitrin. In conclusion, the developed UAE-DES followed by macroporous resin treatment could become an efficient and environmentally friendly extraction and enrichment method for flavonoids from lotus leaves and other natural products.