The invention relates to a preparation method of an SD rat T cell-deficient genetic model, and belongs to the technical fields of genetic engineering and genetic modification. Specific knockout on a key gene Lck, which is in charge of controlling development of rat T cells, is achieved in a rat by virtue of a CRISPR-Cas9 system, so that the SD rat T cell-deficient genetic model is obtained. According to the preparation method provided by the invention, two specific targeting sites, which are targeted to the rat Lck gene, are determined for the first time, and experiments prove that the specific targeting sites are quite high in shear efficiency; two sgRNAs are simultaneously adopted to conduct targeting on the rat Lck gene, and the large-fragment deficient gene knockout rat is obtained, so that in one aspect, the targeted gent completely loses functions, and in the other aspect, subsequent detection is also facilitated. The constructed T cell-deficient SD rat animal model is great significance to immunity and disease researches.