Abstract: Provided are a preparation method and an application of CD7-CAR-T cells. The method comprises: (i) providing a sample to be processed containing T cells, (ii) sorting and activating the T cells contained in the sample, so as to obtain activated T cells, (iii) introducing a first viral vector for expressing a CD7 blocking molecule into the activated T cells, so as to obtain CD7 blocked T cells, and (iv) introducing a second viral vector for expressing a CD7-CAR into the CD7-blocked T cells to obtain CD7-CAR-T cells. By adjusting the transfection sequence and transfection time of the lentivirus expressing the CD7 blocking molecule and the lentivirus expressing the CD7-CAR, transfection efficiency is improved, and the cytotoxicity of the CAR-T cells is enhanced.