Abstract: Objective :To develop monoclonal antibody specific for Cronobacter spp., Seven standard strains purchased from ATCC or DSM were used as antigen. Freeze thawing, ultrasonic smashing, and cell inactivation techniques were applied to prepare antigen. Methods:Spleen cells were collected after common immunization and be used to fuse with hybridoma cell lines sp2/0.20 Cronobacter spp. Strains and 9 non-Cronobacter spp. strains inactivated were served as antigen to screen mAb by Indirect ELISA and Western blot techniques. Results:Five hybridoma cell strains that could secret Cronobacter spp. mAb stably were obtained, appraisal to its subclass, respectively was IgG1, IgG2b and IgG3, isotypes of light strains of the four MAbs all belong toκ. The titers were over 1∶107, and affinity constant was 1.0×1010L/mol. All mAb were specific to few but only few of Cronobacter spp. strains. Conclusion:Cronobacter spp. was a complicated and diversity genus, It is difficult to get one mAb which can cover all species or sub-species, but multi mAb combined use may be an effective way to detect Cronobacter spp. by immunology technique. The established mAb will make a foundation for further research.