A methodology for preparing and certifying the reference material of 3-amino-2-oxazolidinone(AOZ) in eel muscle lyophilisates was presented. Furazolidone was accessed to eel by dipping fish in pond with furazolidone solution at a dosage of ca 0.16 mg/L. With the metabolism of furazolidone in eel, the muscles contain a certain concentration of AOZ as furazolidone metabolite was obtained. Lyophilization of the muscles was performed in one batch and 400 bags of samples were obtained by the procedure of homogenation, cryodesiccation and irradiation. The homogeneity and stability of the sample was examined. The value of the chemical constituent of the sample was certified through the collaborative analysis program participated by 11 laboratories using isotope dilution liquid chromatography-tandem mass spectrometry, and the uncertainty assessment was performed. The reference materials have been approved as certified reference materials by AQSIQ, China (State General Administration of the People′s Republic of China for Quality Supervision and Inspection and Quarantine) in 2009 after one year of trial period. The serial numbers is GBW(E)100180.