Abstract: The biofilm produced by 26 strains of Vibrio vulnificus (Vv) and the factors affecting biofilm formation were studied to provide theoretical basis for effective control of biofilm formation by Vv. In this study, Congo red plate method, improved test tube method and improved microplate method were used to analyze the ability of 25 isolated Vv strains and 1 standard strain to form biofilm, and one strain with the strongest ability to produce biofilm was selected from them. The effects of different initial bacterial concentration, temperature, culture time, pH, NaCl concentration, metal cations and contact materials on the formation of biofilm were investigated. The results showed that 25 strains (96.15%) had biofilm forming ability among the selected strains and VvK had the strongest ability to produce biofilm. At 25 ℃, the initial bacterial concentration was 10⁸ CFU/mL, 3% NaCl and pH8~9 for 24 h, the biofilm quantity was the largest. However, the formation of biofilm was inhibited by certain concentration of metal cations (Cu²⁺, Mn²⁺, Ca²⁺, Mg²⁺) definitely, and the inhibition ability of above cations decreases in turn. When VvK contacted hydrophilic surfaces (stainless steel and glass), the amount of biofilm formation was significantly higher than hydrophobic surfaces (polystyrene), and the amount of biofilm formation on stainless steel was the largest. The biofilm forming ability of different Vv was quite different, and had specific rules under different cultivation conditions, which should be paid more attention.