Objective:To establish a method for the preparation of acetamiprid matrix reference material in cowpeas.Methods:Planted fresh blank cowpea which meets the requirements of reference material development was selected as the research object to obtain dry powder after crushed, freeze-dried, added, mixed, twice freeze-dried, crushed, screened, mixed, packaged and Co60 irradiation sterilized. The dry powder sample was determined by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) and calibrated for quantitative determination by matrix standard solution. After homogeneity and stability studies, coordinated value was determined by 9 laboratories and the uncertainty was evaluated.Results:The prepared sample of cowpea freeze-dried powder had good homogeneity and stability, for which it was convenient to storage transport and preserve in stable status during the testing time. The property value and expanded uncertainty of acetamiprid was (0.283 7±0.008 5) mg/kg (k=2), certified by 9 independent laboratories.Conclusion:The matrix reference material prepared by this method has good homogeneity, stability and accuracy in request of the national metrology technical specification, which can be used in the quality control in process of testing, calibration of analytical instruments and development and verification of testing method, etc.