The invention relates to a preparation method of a milk powder matrix standard substance containing aflatoxin AFTM1. The preparation method is mainly used for calibrating a measuring instrument and evaluating the accuracy of a measuring method. The invention belongs to the technical field of method detection. The method comprises the following steps: obtaining raw milk polluted by AFTM1; removing physical impurities, performing cooling to 3-5 DEG C, and injecting the product into a milk storage tank for storage; performing preheating, and keeping the temperature at 40-50 DEG C without exceeding 60 DEG C; preforming homogenizing, and keeping the homogenizing pressure at 5-20 Mpa; performing sterilizing, controlling the temperature at 80-85 DEG C, and performing concentrating until the solid content is about 40%-50%; performing spray drying, the pressure range of a high-pressure pump being 5-20 Mpa; keeping the temperature of milk powder at an outlet of a fluidized bed at 25-30 DEG C; and performing cooling to room temperature, performing sieving, uniformly performing mixing, performing sealing and sub-packaging. The milk raw material polluted by the aflatoxin AFTM1 is obtained by simulating feeding, and the milk powder sample polluted by the AFTM1 in actual detection is completely simulated by the procedures of impurity removal, homogenization, spray drying and the like, so that the accuracy and the reliability of a detection result are improved.