Human cytomegalovirus (CMV) infection is widespread among adults (60-90%) and is usually undetected in healthy individuals without symptoms but can cause severe diseases in immunocompromised hosts. T-cell receptor (TCR)-like antibodies (Abs), which recognize complex antigens (peptide-MHC complex, pMHC) composed of MHC molecules with embedded short peptides derived from intracellular proteins, including pathogenic viral proteins, can serve as diagnostic and/or therapeutic agents. In this study, we aimed to engineer a TCR-like Ab specific for pMHC comprising a CMV pp65 protein-derived peptide (NLVPMVATV; hereafter, CMVpp65) in complex with MHC-I molecule human leukocyte antigen (HLA)-A02:01 (CMVpp65/HLA-A02:01) to increase affinity by sequential mutagenesis of complementarity-determining regions using yeast surface display technology. Compared with the parental Ab, the final generated Ab (C1-17) showed ~67-fold enhanced binding affinity ( ≈ 5.2 nM) for the soluble pMHC, thereby detecting the cell surface-displayed CMVpp65/HLA-A*02:01 complex with high sensitivity and exquisite specificity. Thus, the new high-affinity TCR-like Ab may be used for the detection and treatment of CMV infection.