Objective To expand the varieties of matrix reference materials, improve the concentration levels of quantitative values, and meet the needs of market detection and scientific research, a matrix reference material for the compositional analysis of zearalenone (ZEN) in whole wheat flour was developed.Methods Naturally contaminated wheat was used as the raw material, and the matrix reference material was prepared through cleaning, milling, and homogenization processes. It was vacuum-packed in aluminum foil pouches and sterilized by irradiation. The effects of grinding particle size and mixing time on the homogeneity of ZEN were investigated. Value assignment was conducted using two methods with different principles: ultra-high performance liquid chromatography (UPLC) and stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS), and was validated through a collaborative study involving seven laboratories.Results The optimal grinding particle size is determined to be 380 μm, with a mixing time of 32 h. The homogeneity test (F-test) and stability studies demonstrate that the reference material remains stable for 7 d at 60 ℃ and for 17 months under light-protected conditions at (20±5) ℃. The ultimate certified value of ZEN in whole wheat flour is determined to be (57.1±6.0) μg/kg (k=2, 95% confidence interval).Conclusion The reference material supplements and improves the matrix reference material system at the limit of mass fraction (60 μg/kg) and can support laboratory method validation, quality control management, and skill assessment of inspectors.