Bacterial biofilm brought great threat in milk preservation. Thus it is very important to research the formation features of biofilm during milk corruption and find an effect way to eliminate the biofilm. The relationship between the biofilm formation ability, the viable count in the biofilm and the AI-2 activity of Staphylococcus aureus, Salmonella and Escherichia coli in the process of milk spoilage were investigated by the method of microtitreplate. The change of biofilm and AI-2 activity after the addition of furanone was tested. Then the gradient concentrations of furanone indicated the relevance between the furanone and the quantity of bateria in the biofilm. The results showed that the sequence of three bacteria for biofilm formation ability, the viable count in the biofilm and the AI-2 activity was Staphylococcus aureus＞Escherichia coli＞Salmonella. Besides, the biofilm formation ability and AI-2 activity both declined after adding furanone, and the biofilm formation ability of Staphylococcus aureus reduced to 25%. Thus the results indicated that the ability of biofilm formation might be regulated by the AI-2 activity which was secreted by the viable isolates in the biofilm.