Objective To prepare proficiency testing samples of Vibrio parahemolyticus （V. parahemolyticus）and apply to laboratory test.Methods The five background strains and V. parahemolyticus were identified by biochemical reaction and matrix-assisted laser desorption ionization time of flight mass spectrometry（MALDI-TOF MS）. The V. parahemolyticus positive samples and negative samples were prepared by freeze-drying. The negative samples contained background bacteria only and the V. parahemolyticus were added in positive samples with background strains. Twenty samples were selected randomly for uniformity test. The samples were stored respectively at -20 ℃ and 4 ℃ to evaluate the storage stability， meanwhile the transport stability were evaluated by storing samples at 25 ℃ and 37 ℃ for 7 d. Samples were distributed to laboratories participating in the proficiency test with operation instruction， and the feedback results from laboratories were analyzed.Results The results of all the related strains were consistent with the expectations. Both of the positive samples and negative samples could meet the requirements of the uniformity and stabilit. The satisfaction rates of assessment results were 87.5% and 90% in 2019 and 2020 respectively.Conclusion The V. parahemolyticus samples could meet the requirements of the proficiency test. By organization proficiency test， the difference of laboratories can be discovered which is helpful to further improve the laboratory test capacity.