Detection of pathogens is of great importance for health and safety. Cronobacter which belongs to common human opportunistic foodborne pathogen can lead to meningitis and necrotizing enterocolitis in infants. So a simple and rapid immunochromatographic strip for detecting Cronobacter was being more in need of and setted up for the first time in our study. CP783 protein was expressed and used as the specific antigen of Cronobacter to obtain polyclonal antibody (pAb) for immunochromatographic strip. Through cloning, expression, purification and animal immunization, we obtained pAb against CP783 protein, which were then conjugated to the fluorescent microsphere as the capture reagent at the test line. Meanwhile, cross reaction with Vibrio parahaemolyticus PVPA 0146 and Salmonella typhimurium ATCC 13311 determined by dot blot were eliminated after purification of pAb against Cronobacter whole cell protein. Afterwards, the pAb against whole cell protein was sprayed as the detective reagent. Our developed fluorescent microsphere-based immunochromatographic strip had high specificity and sensitivity. It resulted in reaching the minimum detectable concentration of Cronobacter at 105 CFU/ml in pure culture and 106 CFU/ml in milk.