Podophyllotoxin, along with its congeners and derivatives exhibit strong biological activity mainly as antitumor drugs and antiviral agents. Here, the ultrasound-assisted extraction of podophyllotoxins including α-peltatin, podophyllotoxin, podophyllotoxinone and deoxypodophyllotoxin from Juniperus sabina L. leaves using deep eutectic solvents (DESs) were developed first time. To simultaneously and effectively extract four target podophyllotoxins, 19 DESs were screened to determine the best solvent to extract podophyllotoxins. Choline chloride-lactic acid (1:1) with 30 % water content was proved to have the best extraction effect. The optimal extraction process with DES were DES/solid ratio 30 mL/g, reaction temperature 44℃, time 53 min and ultrasound power 495 W. Under optimized extraction conditions, the total podophyllotoxins extraction yield was 27.520 mg/g, 196.84 % higher than that using traditional methanol solvent. The yield of α-peltatin, podophyllotoxin, deoxypodophyllotoxin and podophyllotoxinone were 13.567, 9.548, 2.021, 2.204 mg/g, respectively. Subsequently, the podophyllotoxins were effectively isolated from the deep eutectic solvent using D101 macroporous resin. The optimal process for enriching target podophyllotoxins by D101 macroporous resin were as follows: 30 mg/mL of crude extract solution, five bed volumes feed volume for adsorption; six bed volumes of 80 % ethanol for desorption. Moreover, a scale-up enrichment experiment of podophyllotoxins was performed under optimum procedure, and the results showed that the target podophyllotoxins yield was increased by 8.92 times, with recoveries in the range of 75.86–85.69 %. Additionally, the enriched podophyllotoxins effectively restored the antibacterial efficacy of cefazolin against methicillin-resistant Staphylococcus aureus and colistin-resistant Escherichia coli, resulting in 16–64-fold reduction in MIC. Therefore, the method developed in this study could be an eco-friendly, efficient and sustainable alternative for the selective extraction and enrichment of active compounds from plant material.