The formation condition of Cronobacter sakazakii biofilm and its elimination by ultrasound treatment on glass surfaces were investigated through biofilm establishment in vitro model by 96/6 orifice culture method and turbidimetric determination of biofilm content. The morphology of biofilm was observed by crystalline violet staining. The ultrasonic shedding was evaluated by plate colony counting method and the elimination effect of ultrasonic treatment was observed by laser scanning confocal microscopy. The optimal temperature and pH for Cronobacter sakazakii formation was 37 ℃ and 7.5. NaCl (2.5%), CaCl2(0.50%) and MgCl2(1.50%) added in medium promoted biofilm formation obviously, Cu2+(1.50%) and EDTA(2.5%) added in medium inhibited bioflm formation. Cronobacter sakazakii biofilm attached to the glass surface was eliminated by ultrasonic treatment. The elimination effect was the best when initial temperature was 30 ℃ and time was 14 min. The change of living cells number on biofilm after ultrasound treatment was observed by laser scanning confocal microscopy to prove and evaluate the elimination effect of ultrasonic treatment.