The invention discloses a preparation method and application of a targeting mesenchymal stem cell (MSC) drug delivery system. The preparation method comprises the steps as follows: S1, stem cell collection; S2, cell detection; S3, production of a transfection mixed solution; S4, pretreatment of MSCs; and S6, alkaline culture. The MSCs are widely sourced, can be xenogenous as well as autogenous andhave sufficient sources, and the cells have excellent capacity in proliferation, survival, differentiation, migration, synthesis, factor secretion and other aspects, are free of ethical problems andhave low immunogenicity and broad application prospects. The disclosed tissue damage targeting MSCs can be effectively planted at tissue damage parts, blood washing or natural falling can be avoided,the homing cell quantity can be increased, besides, low expression of smad protein can be avoided, and the fibrosis promotion risk due to transplantation of the MSCs can be avoided.