The invention belongs to the technical field of a standard sample of an animal-sourced substrate drug in detection of veterinary drug residue and discloses a preparation method of a freeze-dried powder sample of toltrazuril in chicken meat and a toltrazuril sulphone residue. The method includes following steps: feeding chicken with the toltrazuril; enabling the toltrazuril and a metabolite toltrazuril sulphone to remain in chicken after toltrazuril sulphone with time controlling; killing the chicken to obtain breast muscle; performing a skin removing and blood draining process; adding a vitamin C solution; performing a homogenizing process to obtain meat paste; performing a vacuum freeze-drying process and a crushing and sieving process; performing a nitrogen-filling packaging process in an anaerobic work station; performing a vacuum packaging process to a polyethylene bottle with an aluminium foil bag after a sealing process being carried out to obtain a standard sample of the freeze-dried powder. The sample can be transported at normal temperature and is long in preserving time. A situation of combination of a target object and the substrate is coincided with that of an actual detection sample. The sample can be used of quality controlling of relative detection and analysis, is homogeneous and stable, is convenient to transport, is reliable in use and has significant economic value and market competitiveness.