Abstract: In order to investigate Cronobacter sakazakii.biofilm ( 48 h) on the surface of glass slide, polypropylene and stainless steel, the fluorescence image stacks of C.sakazakii biofilm tagged with SYTO 9/PI was assayed with hyphened technique of the program confocal laser scanning microscopy ( CLSM) and COMSTAT. The result showed that the sequence from high to low of total biomass, average thickness and average diffusion distance was: stainless steel, polypropylene and glass slide, while the sequence from high to low of roughness coefficient and surface to volume was: glass slide, polypropylene and stainless steel.Live/dead ratio of C.sakazakii biofilm on the surface of glass slide was higher than polypropylene and stainless steel.Compared with strain Ⅰ and Ⅱ, the biofilm of C.sakazakii strain ATCC 29544 had higher level of total biomass, live/dead ratio, average thickness and average diffusion distance, while roughness coefficient and surface to volume had lower level. In conclusion, C.sakazakii produced stronger biofilm on the surface of stainless steel than glass slide and polypropylene, and the biofilm forming ability of strain ATCC 29544 was higher than strain Ⅰ and Ⅱ isolated from food.