In this study, an ultrasonic assisted natural deep eutectic solvent (DES) was used to extract hydroxytyrosol (HT) from olive leaves. The optimal extraction conditions of the MaPa-4 concentration, extraction time and solid–liquid ratio were obtained by single factor experiments. The formation mechanism of MaPa and its interaction with HT were analyzed by FTIR, ¹H-NMR and density functional theory (DFT) calculation. Then, MaPa-4 and water extracts obtained under the optimal extraction conditions were selected for a series of efficacy tests. MaPa-4 extract demonstrated low cytotoxicity, good biocompatibility, and excellent anti-inflammatory and bacteriostatic properties. Overall, MaPa-4, as an environmentally friendly and efficient solvent, was combined with ultrasound treatment to develop an efficient, green and feasible method to extract HT from olive leaves.