Objective To prepare Cronobacter spp. (Enterobacter sakazakii) proficiency test samples which meet the requirements of uniformity and stability, and use them for proficiency test. Methods The background strains and Cronobacter. spp were identified by biochemical and matrix-assisted laser desorption ionization time of flight mass spectrometry method. Freeze-drying was used to prepare the samples with 10⁴CFU/bottle. According to Guidance on Evaluating the Homogeneity and Stability of Samples Used for Proficiency Testing, 20 samples were selected randomly for uniformity test. The samples were stored at -20 and 4 ℃ respectively to evaluate the storage stability, and at 25 and 37 ℃ to evaluate the transport stability. According to GB 4789.40—2016, samples were distributed to participating laboratories with operation instruction, and then the feedback result were analyzed. Results The identified result of all the related strains were correct and consistent with expectations. The result of uniformity and stability tests met relevant requirements. All of the experiment result submitted by 25 laboratories within the required time were correct, and the satisfaction rate was 100%. Conclusion The samples of Cronobacter spp. Enterobacter sakazakii in milk powder proficiency test can meet the relevant requirements, and this proficiency test provides external quality control assessment result for the participating labratories simultaneously.