Abstract: Modified microtiter-plate test was used to investigate the biofilm formation of 114 Vibrio alginolyticus strains. The strongest biofilm formation strain was selected for biofilm characteristics study under various environmental factors. Further,the biofilm formation was observed by using fluorescence microscopy. The results showed that among all the tested strains,112(98.25%) had biofilm formation ability,and 65(57.02%)were at the strong or moderate level. V. alginolyticus formed strong biofilm in p H6.00⁸.00 TSB with 3% Na Cl at 28 ℃ for 16 h. The ability of biofilm formation was inhibited by adding a certain concentration of Cu2+,Mn2+,Ca2+,Mg2+. It's also found that the bacteria adhered to hydrophilic surfaces(stainless steel and glass) better than to hydrophobic ones(polystyrene),and adhered best to stainless steel sheet. The fluorescence microscope observation was consistent with the modified microtiter-plate results. It concluded that V. Alginolyticus biofilm forming-ability was affected by various environmental factors with diversity among different strains. The results provided a theoretical support for the effective control of V. Alginolyticus biofilm formation.