Abstract: To explore the extraction process of mycosporine amino acids (MAAs) and the antibacterial mechanism of Vibrio parahaemolyticus, the effects of liquid-solid ratio, extraction temperature and extraction time on the yield of MAAs were analyzed by single factor and response surface optimization tests, and MAAs were isolated and identified by silica gel column chromatography, HPLC and ESI-MS. The antibacterial mechanism of MAAs against Vibrio parahaemolyticus was analyzed by scanning and fluorescence microscopy to observe the leakage of intracellular nucleic acids and proteins, and DNA decomposition. The results showed that the optimal extraction parameters of MAAs of Ulva lactuca was liquid-solid ratio of 25 mL/g, extraction temperature of 40 ℃ and extraction time of 118 min, and the yield of MAAs was 179.34±3.91 mg/g. MAAs significantly destroyed the cell membrane of Vibrio parahaemolyticus, causing the leakage of nucleic acid and proteins, and promoting the breakdown of DNA in the bacteria. MAAs were isolated by silica gel column chromatography to obtain two fractions, and the fraction H2 was identified as shinorine, and the content in Ulva lactuca dry powder could reach 10.32 mg/g. This study provides a technical reference for the preparation of MAAs of Ulva lactuca and the biological control of Vibrio parahaemolyticus.