Mycosporine-like amino acids (MAAs) are a family of intracellular ultraviolet (UV) -absorbing compounds involved in the protection of aquatic organisms against solar UV radiation. The protocols used to extract MAAs largely differ; for example, differences include the type of solvent, concentrations, temperatures, and times used. Since ethanol is less expensive and less toxic than methanol, the procedure used in this study to extract the MAAs of Gracilaria tenuistipitata was based on ethanol. An optimization study of G. tenuistipitata MAA extraction using response surface methodology (RSM) was performed, and 3D response surfaces were plotted from the mathematical models. The optimal conditions based on combination responses were as follows: a temperature (X1) of 63℃, a time (X2) of 165 min, an ethanol concentration (X3) of 74.3% ethanol. Under the optimum extraction procedure conditions, the yields of crude MAAs and MAAs were 4.1 mg/g and 0.83 mg/g dry weight. Additionally, crude MAAs of G. tenuistipitata significantly increased cell proliferation and protected against UVB-induced damage in HS68 fibroblast cells. Our study revealed that crude MAAs of G. tenuistipitata present a potential source of natural photoprotective compounds.