CRISPR/Cas9-based genome editing enables precise DNA insertion at defined loci and represents a promising platform for T cell immunotherapy. Targeted TCR knock-in (KI) enhances antigen specificity; however, efficient KI in human primary T cells remains challenging due to activation dependency and low homology-directed repair (HDR) efficiency. In this study, we optimized a CRISPR/Cas9-based KI protocol for human primary T cells under serum-free conditions using a plasmid donor template to facilitate HDR-mediated insertion of the target TCR sequence. The protocol enabled simultaneous knockout (KO) of the endogenous TCR gene and precise insertion of the NY-ESO-1-specific TCR 1G4 sequence, including the Vβ13.1 chain, into the target locus. By optimizing cell activation and electroporation-based RNP delivery conditions, the TCR was successfully introduced into the genomic locus, and stable surface expression confirmed efficient knock-in of the Vβ13.1 chain. Furthermore, CD62L and CD45RA analysis showed that approximately 50% of edited T cells retained a central memory phenotype after gene insertion. Additional optimization of donor DNA and RNP conditions further improved overall KI efficiency. In conclusion, HDR-based TCR knock-in efficiency can be substantially improved under serum-free conditions while preserving T cell differentiation stability, supporting the development of clinically applicable TCR-T cell manufacturing strategies.