In this study, ultrasound-assisted extraction was performed to extract ascorbic acid from rugosa rose (Rosa rugosa Thunb.) fruit. The optimal conditions were investigated by response surface methodology, using two variable including reaction time (16-44 min) and temperature (16-44°C). The ascorbic acid extraction was sensitive to the reaction time rather than the reaction temperature, and the optimal conditions for ascorbic acid extraction were 25°C and 30 min. Ascorbic acid and gallic acid in the rugosa rose fruit extract were completely separated by HPLC, with a resolution factor of over 1.5 between the two. The correlation coefficient of the ascorbic acid was 0.999 in a linearity test for 50-150 μg/mL concentration of extract. The limit of detection and limit of quantification values were 0.16 μg/mL and 29.89 μg/mL, respectively. The relative standard deviations (RSD) for repeatability and reproducibility were determined, and each RSD showed good precision at less than 5% (N=6).