The objectives of this study were to investigate the growth characteristics of Salmonella typhimurium biofilm（BF） on glass surface， effect of ethanol activity on the formation of S. typhimurium BF and effect of ethanol adaptation on the resistance of planktonic and BF to lethal stress. The morphology of S. typhimurium BF on the glass surface was observed by crystal violet staining， BF biomass by Thermo Scientific Microplate Reader at 570 nm and ethanol adaptation by plate colony count. Results showed that S. typhimurium BF formed on the glass surface could be directly observed by crystal violet staining， and reticular structure formed by S. typhimurium BF became denser with incubation time. Ethanol activity had significantly different effects on the formation of BF. Ethanol added at different level during 48 h incubation of S. typhimurium inhibited the formation of BF significantly， however， ethanol added after the incubation of S. typhimurium for 24 h at 5% level only promoted the growth of BF. Ethanol at level of 5% increased the tolerance of bacteria to malic acid in BF formation under the condition of 1/10 TSB. In addition， 5% ethanol adaptation increased planktonic resistance to 12% ethanol and 5 mg/mL malic acid.