Objective To prepare proficiency testing samples of Cronobacter spp. and apply to laboratory test in 2018-2020. Methods The background bacteria and Cronobacter spp. were identified by biochemical reaction and MALDI-TOF MS. Cronobacter spp. negative samples (background bacteria only) and positive samples (background bacteria and Cronobacter spp.) were prepared by freeze-drying. 20 samples were selected randomly for uniformity test. The samples were stored respectively at -20 ℃ and 4 ℃ to evaluate the storage stability, at 25 ℃ and 37 ℃ for 7 d to evaluate the transport stability. Samples were distributed to laboratories participating in the proficiency with operation instruction, and the feedback result from laboratories were analyzed. Results The identified result of all the related strains were correct. Both of positive samples and negative samples were uniform and stable. The satisfaction rates of assessment result were 97.1%, 94.7% and 100% respectively in 2018-2020. Conclusion The samples could meet the requirements of proficiency test. By organization proficiency test, the difference of laboratories can be reflected which is helpful to further improve the laboratory test capability.