Abstract: Marine macroalgae is one of the good sources of mycosporine-like amino acids (MAAs), especially red macroalgae. As a new type of active substances with commercial development prospect, extraction, isolation and characterization of MAAs are far from enough for the application. To obtain the extraction process of MAAs from four species of red macroalgae (Bangia fusco-purpurea, Gelidium amansii, Gracilaria confervoides, and Gracilaria sp.), a series of single-factor and orthogonal experiments were carried out to analyze the effects of extraction temperature, time, times and solid-liquid ratio on the yields of MAAs. The extraction process of MAAs was optimized, and the extracts were isolated and identified. The optimized extraction processes of MAAs were obtained as follows: 45 ℃, 3 h, 4 times, 1:25 g/mL. 45 ℃, 1 h, 4 times, 1:20 g/mL. 45 ℃, 2 h, 3 times, 1:15 g/mL. 40 ℃, 1 h, 3 times, 1:20 g/mL. Four MAAs extracts of red algae were prepared by using the above extraction process, and the yields were 249.3, 197.9, 146.4 and 449.5 mg/g, respectively. All of them showed UV absorption characteristics and color reaction of MAAs in UV spectrum and thin layer chromatography (TLC). Furthermore, MAAs extracts from Bangia fusco-purpurea (or Gracilaria sp.) were isolated by silica gel column chromatography to obtain one fraction H1 (or two fractions J1 and J2). MAAs composition and proportion of these fractions were determined by UV, HPLC and MS, and comparison with existing literatures. H1 included shinorine, palythine and porphyra-334 (the content of these three MAAs was 95.4% of the content of the fraction H1), and palythenic acid (4.6%). Shinorine, palythine, porphyra-334 (96.3% of the fraction J1), and palythenic acid (3.7%) were in J1. And J2 was palythine.