Background: Thalassia hemprichii (Ehrenb.) Asch is one of the commonly found seagrasses spread over the coast of Indonesia and has not been utilized. Previous studies have reported its potency as an antioxidant, however, studies on optimal extraction conditions using ethanol as a solvent to obtain higher antioxidant activity are still limited. Objective: The purpose of this study is to optimize the extraction condition for obtaining the highest antioxidant activity and total phenolic content of the seagrass T. hemprichii. In an addition, percentage yield of extract was also evaluated. Methods: In this study, factorial design with independent factors of ethanol concentrations (50%, 70%, and 100%), the use of HCl 1 N (Yes/No), and temperature (300C, 600C) was developed. The analysis of variance (ANOVA) was used to determine the significance of the above factors towards antioxidant activity and total phenolic content. The antioxidant activity was evaluated with 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and the total phenolic content was determined by Folin-Ciocalteu method. Results: The highest antioxidant activity (IC50=83.48 μg/mL) was shown in sample R1 (extracted with 50% ethanol, added with HCl 1 N, and 600C). The sample was also showed total phenolic content 41.03 mg GAE per gram and 22% yield extract. Among the factors examined, the used of HCl 1 N showed the most significant effect to the antioxidant activity and the total phenolic content, with the p-value of 0.048 and 0.034 respectively (p<0.05). Conclusion: This extraction condition can be applied to obtain higher antioxidant activity and total phenolic content from the plant.