Abstract: Objective To develop proficiency testing samples for pathogenic bacteria in cosmetics, then to set up an effective sample preparation process, and establish a scientific and reasonable evaluation basis. Methods The medium ingredients for proficiency testing samples of cosmetics were determined by screening experiment, and then prepared as cosmetic solution. After sterilization, the medium solution was mixed with testing objects (testing indicator bacteria and related interference bacteria in cosmetics), and dispensed into freeze-dried glass tubes. Next, the mixture was sublimated and crystallized by freezing and drying technology, then testing samples were vacuumed and encapsulated. Finally, inter-laboratory comparisons were conducted after uniformity and stability tests of the proficiency testing samples to evaluate the laboratory testing capabilities and validate expected results. Results Six cosmetics (Skim milk powder, Trehalose, Glycerol, Vaseline, Tween-80, and Methoxy ethyl cinnamate ethyl ester) were determined as medium ingredients for proficiency testing samples, then mixed with testing objects. The sampling results of 36 samples to be identified by sampling ratio 10% showed that the proficiency testing sample had good uniformity. The stability tests were proceeded at refrigerated storage temperature, room temperature (25 to 28 ℃), and raising temperature. The data showed that the significant change was not observed in the bacteria amounts in proficiency testing sample within 35 days at 4 ℃ and room temperature (all P>0.05). At 37 ℃ and 42 ℃, the amount of bacteria decreased quickly. However, the samples would be stable for 16 days or less at 37 ℃, and 12 days or less at 42 ℃. The inter-laboratory comparisons by 21 comparison labs showed that the satisfaction was 90.5% and basic satisfaction was 9.5%. Conclusion The preparation of freeze-drying proficiency testing samples in this study is suitable for proficiency testing of pathogenic bacteria in cosmetics.