Biofilm is a structured community of microorganism encapsulated within a self-developed polymeric matrix and adherent to a living or a solid surface. In this study, we investigated the effects of various substrates on the formation of biofilm in Vibrio parahaemolyticus. We found that biofilm formation profoundly increased in a substrate, that consisted of calcium chloride, calcium nitrate, and calcium sulfate in 1% peptone water. On the other hand, a dramatic reduction in biofilm formation was observed in a substrate, that consisted of glucose and ferric chloride in LB broth. These results suggest that V. parahaemolyticus prefer to form a biofilm on the surface of a crustacean or a clam, where calcium ion is rich, and also where seawater temperature is relatively lower. In contrast, high levels of glucose in a crustacean or a clam body resulting from increased seawater temperature, can make V. parahaemolyticus detach from it and lead to free floating.