Polyphenols are phytochemicals that exist in grapes and are beneficial to human health. In this study, resveratrol, oxyresveratrol, and piceatannol in wine were extracted by deep eutectic solvent dispersive liquid-liquid microextraction (DES-DLLME), and a method was established for quantifying these polyphenols by high-performance liquid chromatography-UV/Vis (HPLC-UV/Vis). Several parameters pertaining to sample extraction, clean-up, and concentration were optimized and verified with central composite design (CCD) using Design Expert 11. The optimized sample preparation parameters are as follows: the DES extraction solvent, tributylmethylammonium chloride/decanoic acid (1:3 M ratio); basic solvent, 1.3 mL of 5% potassium bicarbonate; volume of acetic anhydride, 250 μL; derivatization time, 5 min; dispersive solvent, methanol; ratio of extraction and dispersive solvents, 1:5.5; and salt, 1.0 g. Chromatographic separation by HPLC/UV-Vis was performed on an ACME C18 (4.6mm id × 150 mm length, 5 μm particle size) column in gradient elution mode using water and 70% methanol. Under the established extraction and HPLC-UV conditions, the limit of detection (LOD) and limit of quantitation (LOQ) of the three analytes in spiked samples ranged from 1.69 to 2.53 μg/L and 5.64 to 8.42 μg/L, respectively. Recovery studies were performed in low, medium, and high concentration ranges to establish a calibration curve, and the accuracy and precision in the working range were 95.1-108.0% and 1.3-6.7 RSD%, respectively. The calibration curves for quantitative analysis were obtained in the concentration ranges 5.6-56.4, 8.3-82.6, and 8.4-84.2 μg/L, with correlation coefficients (r²) ranging from 0.9947 to 0.9967. The proposed method was applied to the determination of polyphenols in wine samples.