The invention provides a preparation method of chimeric antigen receptor T cell of targeted CD19, and relates to the technical field of gene engineering. The method comprises the following specific steps: construction of CD19-CAR lentiviral vector, preparation of lentivirus, separation of T cell and infection and proliferation of T cell. The method optimizes a transfection system and a culture medium adopted during cell culture. The method improves the transduction efficiency of T cell, solves the problem of insufficient transfection efficiency, obviously improves the kill capability of CAR-Tcell to CD19 positive cell, ensures that the kill capability reaches greater than 98 percent, and shows huge potential in cancer treatment.