A method for preparing NY-ESO-1-specific TCR. A fruit fly antigen presenting cell is loaded with NY-ESO-1 antigen peptide and activated CD8+T cells to produce CTL cells. The cytotoxic CTL cells are then stained with HLA-A*02:01 NY-ESO-1 Tetramer-SLLMWITQC-APC (MBL, TS-M011-2) and CD8-FITC fluorescent double staining, and the flow cytometry assay is used to screen and collect dual positive CTL cells. The selected CTL cells are then subjected to single-cell TCR gene sequencing to obtain TCRα and β variable region gene sequences. The TCR genes are then optimized and designed, and the constant region of the TCR genes is converted to a murine source and supplemented with signal peptides to obtain the full gene sequence of TCRα and β chains. TCR α and β chains are genetically synthesized and connected with the IRES sequence to ensure that TCRα and TCR β chains are co-expressed in a plasmid to obtain TCR genes. The TCR genes are then expressed in eukaryotes.