Natural deep eutectic solvent (NaDES) is generally considered as a greener alternative to fossil solvent, with great potential in various areas. In the present work, 25 different NaDESs were screened for the extraction of puerarin (PUE) and its two natural derivatives from Radix Pueraria (RP). As the main isoflavone in RP, PUE has a wide range of biological activities. However, its application is restricted due to its poor solubility in water and low oral bioavailability. In this study, the extraction of PUE with NaDESs showed significant advantages compared with traditional solvents. While using L-Pro-Maa (L-proline-malic acid) under optimal conditions, the optimized yields of PUE, 3-MPR and PRX were 98.7 mg/g, 16.3 mg/g and 9.9 mg/g, respectively, which were 2.2-, 2.9- and 3.4-fold higher than that of water. Furthermore, the oral bioavailability of PUE in NaDES extracts was comparatively investigated in rats with HPLC-MS technique. Pharmacokinetic analysis revealed that the relative bioavailability of PUE in L-Pro-Maa extract is 323%. The result indicated that NaDES is not only a sustainable ionic liquid with higher extraction efficiency, but also an enhancer of oral bioavailability of specific natural products.