A method for preparing and certifying the reference materials of toltrazuil and toltrazuil sulfone in chicken muscle lyophilisates was presented.Toltrazuil was accessed to chicken by oral administration.With the metabolism of toltrazuil in chiken,a certain concentration of toltrazuil and toltrazuil sulfone were obtained in the muscles.Lyophilization of the muscles was performed in one batch,and 852 bags of samples were obtained by the procedure of homogenation，cryodesiccation and vacuum-packaging.The homogeneity and stability of the sample were examined by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).The value of the chemical constituent of the sample was certified through the collaborative analysis program participated by 8 laboratories,and the uncertainty was evaluated.The standard values of standard samples were certified as(60.42±9.12)μg/kg for toltrazuil and (73.10±12.48)μg/kg for toltrazuil sulfone.The results showed that good homogeneity and stability of the sample reached up to 24 months,and could be used for the quality control of toltrazuil and toltrazuil sulfone residue in chicken.