The invention relates to a preparation method of an antitumor drug screening model with 4.1R and CD29 as target spots and application thereof, wherein a protein 4.1R gene knockout mouse is used as experimental material for preparing C57BL/6J_4.1R-/-MEF primary cells, and the SV40 large T antigenic conversion method is used for obtaining immortalized C57BL/6J_4.1R-/-MEF. The invention aims at providing an in vitro cell screening model for new drug screening and gene therapy by using protein 4.1R and CD29 as the drug targets. The invention has the advantages that the limit of drug screening and production by using 4.1R as the treatment target owing to high raising cost of gene knockout animals and in vitro passage limitation of the primary cells can be avoided and the production cost of the new targeted drug is lowered. The activation of CD29 on the cell surface is inhibited by the lack of 4.1R in the built C57BL/6J_4.1R-/-MEF, and the screening model is used for producing and researching the new antitumor drug related to cell proliferation and migration, cell factor secretion, cell signal transduction and regulation and tumor drug resistance with CD29 as the target.