Deep eutectic solvents (DESs), a new group of ecofriendly solvent combined with the ultrasonic-assisted extraction (UAE) technique, were first successfully used for extraction of resveratrol from peanut roots. Resveratrol in the extract was analyzed and quantified using a HPLC-UV method. A series of DESs consisting of choline chloride (ChCl) and 1,4-butanediol, citric acid, and ethylene glycol were formulated, finding ChCl/1,4-butanediol was a most proper extraction system. The optimal extraction parameters were obtained using a Box–Behnken design (BBD) test combined with response surface methodology as follows: 40% of water in ChCl/1,4-butanediol (1/3, g/g) at 55°C for 40 min and solid/liquid ratio of 1:30 g/mL. The total extraction content and extraction yield of resveratrol from peanut roots could reach 38.91 mg/kg and 88.19%, respectively, under such optimal conditions. The present study will provide a typical example for using DESs to extract natural bioactive compounds from plants.