Objective: A method for the preparation and determination of amino-mebendazole matrix reference material in eel muscle powder was established. Methods: A positive sample was obtained by culturing eels with mebendazole and was prepared into amino-mebendazole matrix reference material, which went through freeze-drying, vacuum packaging, and radiation sterilization in turn. And its homogeneity, stability, cooperative setting value, and uncertainty were examined. Results: This matrix reference material has good uniformity and can adapt to low temperatures, normal temperatures, long-distance transportation, and other environments. It can be stored for more than 12 months under -18 ℃. The characteristic value of amino-methylimidazole is (35.3±6.4) μg/kg(k=2) through the collaborative setting of 8 qualification inspection institutions. Conclusion: This matrix reference material meets the technical requirements of national standard samples and can meet the needs of quality control and method validation for amino-mebendazole residue detection in eel muscle. This study can provide a good reference for the development of food matrix reference materials.