Amomum villosum Lour. (A. villosum) is widely utilized in culinary and medicinal contexts due to its rich diversity of bioactive compounds. In this study, an ultrasonic-assisted extraction (UAE) method using a natural deep eutectic solvent (NaDES) was employed to extract flavonoids from A. villosum. The NaDES composed of betaine and glycerol (Bet-Gly) exhibited effective performance as an extraction solvent, as evidenced by its high efficiency. Optimal extraction conditions (water content: 30 %, solid-liquid ratio: 1:20 g/mL, temperature: 70 °C, ultrasonic power: 85 % P, time: 30 mins) were established via single-factor experiments. The maximum total flavonoid content (TFC) of 82.22 ± 0.39 mg rutin equivalent per gram dry weight (mg Rut/g DW) was achieved under optimized conditions. Comparative studies revealed that the UAE-NaDES method afforded higher TFC and DPPH radical scavenging activity values, broader flavonoid diversity, and demonstrated a better capacity to stabilize them compared to conventional solutions. Furthermore, NaDES exhibited favorable biocompatibility properties, and the resulting extract displayed remarkable anti-inflammatory and anti-cancer effects. These findings demonstrate that NaDES is an efficient solvent for flavonoids extraction from A. villosum, with potential applications in food and pharmaceutical industries, thereby minimizing solvent removal steps.