Because of lacking of domestic fusarium mycotoxins reference materials for quality control, the certified reference materials (CRM) of deoxynivalenol was prepared and certificated.Methods According to the requirements of JJG 1006-94, the wheat flour samples were homogenized, packed and sterilized. Homogeneity, stability studies and assignment of the certified mass fraction were based upon an in-house study using high-performance liquid chromatography isotope-dilution tandem mass spectrometry. Simultaneously, an inter-laboratory comparison study involving eight laboratories was conducted in order to support the in-house certification study. Results Three samples from 16 randomly selected packages were quantitatively analyzed. Shelf life at a storage temperature of 25 ℃ for one year (long-term stability) or 44 ℃ for 14 days (short-term stability) was considered. The data were evaluated by ANOVA test. There was no significance of homogeneity and stability. The certified values and the corresponding uncertainty was (2 366±187) μg/kg.Conclusion The natural reference material was produced with a certified deoxynivalenol in wheat flour showing relevant mass fractions. The intended purpose of this reference material, for deoxynivalenol, is not only attributed to laboratory internal quality control but also in facilitating the validation of new analytical methods.