In this work, a novel three-constituent natural deep eutectic aqueous solution (3c-NADES) composed of betaine, triethanolamine, and MgCl 2 •6H 2 O was synthesized and used for extracting three main kinds of active compounds from Acanthopanax senticosus stems with a microwave-ultrasoundassisted technique. The target analytes were determined by an ultraperformance liquid chromatography-tandem triple-quadrupole mass spectrometry (UPLC-MS/MS) approach. Single-factor experiments and Box−Behnken design were used to obtain the optimized extraction conditions: a microwave power of 600 W, an extraction temperature of 59 °C, an ultrasound power of 500 W, an extraction time of 42 min, and a solid−liquid ratio of 0.07 g/g. The yields of isofraxidin, eleutheroside E, eleutheroside B, protocatechuic acid, chlorogenic acid, and ursolic acid reached 18.76, 49.38, 174.82, 2.98, 457.39, and 16.62 μg/g, respectively, which increased from 1.04-to 3.19-fold from those of conventional extraction methods. In addition, the reusability of 3c-NADES was stable after six runs. Furthermore, the results with molecular dynamics combined with density functional theory analyses revealed that this kind of 3c-NADES is compatible with the target compounds, and the changes of the hydrogen-bond-related parameters were analyzed. In conclusion, this extraction method is suitable for highly efficient extraction and enrichment of natural active compounds from plant materials.