Abstract: To explore the effect of mycosporine-like amino acids (MAAs) extracted from Sargassum sp. on the activity of tyrosinase, this study first employed single-factor and response surface optimization experiments to analyze the effects of several factors such as methanol concentration, liquid-to-solid ratio, extraction temperature and extraction time on the extraction of MAAs from Sargassum sp.. An optimal extraction process for MAAs from Sargassum sp. was then established using the yield of MAA extracts and their inhibitory rate against tyrosinase activity as indicators. Further, thin-layer chromatography, ultraviolet spectroscopy, high-performance liquid chromatography, and spectrophotometry were used to conduct qualitative and quantitative analysis, in vitro antioxidant analysis, and anti-tyrosinase activity analysis of MAA extracts. And then, the inhibition mechanism of MAAs from Sargassum sp. on the activity of tyrosinase was determined through enzyme reaction kinetics analysis, copper ion chelation experiments, and tyrosinase fluorescence quenching experiments. The results showed that the appropriate extraction parameters for MAAs from Sargassum sp. were a temperature of 41 ℃, a liquid-solid ratio of 26 mL/g, a time of 2.6 h, and a methanol concentration of 31%. The yield of MAA extracts was 89.87±0.73 mg/g, and the IC₅₀ (Half maximal inhibitory concentration) value of its inhibitory rate on the activity of tyrosinase was 1.091±0.32 mg/mL. MAA extracts from Sargassum sp. exhibited the capacity to scavenge DPPH (IC₅₀ of 0.17±0.06 mg/mL) and ABTS (IC₅₀ of 1.03±0.21 mg/mL) free radicals, and could chelate copper ions (with a half-chelation concentration of 0.70±0.21 mg/mL). The inhibitory effect of MAAs from Sargassum sp. on the activity of tyrosinase was a reversible and mixed-type inhibition. MAAs from Sargassum sp. possess the inhibitory activity against tyrosinase and can effectively block the melanin synthesis pathway. They have potential application prospects in preventing the enzymatic browning of fresh fruits, vegetables and beverages, as well as in preventing melanosis in shrimps.