Abstract: The biofilm explored the formation process of E. coli in the calf diarrhea. The experiment used the 96-well micro plate method screened and counted biofilm in 60 strains in E. coli from calf diarrhea fecal which were separated and identified in laboratory. The results showed that get nine positive biofilms in E. coli and determined the positive biofilm growth curve in E. coli. The test concluded and known the biofilm formation period contained four stages: initial adhesion, adhesion, mature biofilm and shedding processes, and then in different biofilm formation process varied in E. coli. The experiment was aimed of providing a new theoretical basis for the treatment against calves diarrhea in clinical.