The production of tetrodotoxin (TTX) using Vibrio sp. YE-101, a novel marine microorganism isolated from the intestine of pufferfish, was investigated. Culture condition was optimized for the enhanced production of TTX using response surface methodology. The experimental sets of environmental conditions including pH, temperature and NaCl concentration were designed using central composite experimental design. The optimal conditions of pH, temperature and NaCl concentration were determined to be 8.1, 29.2℃, and 2.6% (w/v) respectively. The relative growth extent could be enhanced up to 80%, and final mouse unit (MU) value of TTX was also enhanced up to 87% by response surface optimization.