Ginkgo (Ginkgo biloba L.) is one of the oldest trees on earth and its leaves are traditionally used as herbal medicine. The beneficial effects of ginkgo leaves are attributed to the presence of flavonoids and terpene trilactones. Monomeric flavonoids are frequently studied, but recently much attention has been paid to biflavonoids, dimeric flavonoids, due to their biological activity and potential pharmaceutical use. The development of a suitable and effective extraction protocol focusing on biflavonoids is increasingly in demand. Therefore, in this study, we developed an optimized extraction protocol using deep eutectic solvents (DESs) to extract flavonoids from ginkgo leaves, with special attention to biflavonoids. Initially, 250 DESs were screened using COSMOtherm software to identify those that could strongly solubilize biflavonoids and flavonoids, and the results obtained indicated 15 DESs. Among them, betaine: ethylene glycol (B:EG) 1:2 with 30 % H2O (w/w) showed higher extraction efficiency than the extraction performed with 80 % methanol. In a next step, the extraction with the selected DES was further optimized using a Box-Behnken experimental design. The results obtained showed that the optimum extraction conditions using the selected DES were as follows: Extraction time 45 minutes, the ratio of the initial mass of the shredded leaves to the volume of the DES 20:1 and the temperature 20 °C. Finally, the concentrations of the 5 most abundant biflavonoids in ginkgo leaves (amentoflavone, ginkgetin, isoginkgetin, bilobetin and sciadopitysin) in the extracts were determined using HPLC-DAD. The results showed that B:EG 1:2 with 20 % H2O (w/w) and 30 % H2O (w/w) were suitable for the extraction of all five biflavonoids.