The study introduces VADER2.0, an enhanced virus-assisted directed evolution strategy for improving tRNA mutants in mammalian cells. This method overcomes the limitations of previous VADER by processing larger mutant libraries and employing a novel library design that maintains base-pairing between distant residues. The optimized VADER2.0 enabled simultaneous engineering of the entire acceptor stem of *M. maezi* pyrrolysyl tRNA (tRNA^Pyl), leading to the identification of a highly active variant, PyOtR. PyOtR significantly improves the incorporation of various noncanonical amino acids (ncAAs) and facilitates the development of stable cell lines expressing ncAA incorporation machinery. The enhanced activity of PyOtR is demonstrated through improved ncAA incorporation efficiency, higher suppression efficiency of nonsense codons, and better performance in viral vector construction and stable cell line generation. These findings highlight the potential of VADER2.0 for expanding the capabilities of directed evolution in mammalian cells and advancing the technology of ncAA mutagenesis.The study introduces VADER2.0, an enhanced virus-assisted directed evolution strategy for improving tRNA mutants in mammalian cells. This method overcomes the limitations of previous VADER by processing larger mutant libraries and employing a novel library design that maintains base-pairing between distant residues. The optimized VADER2.0 enabled simultaneous engineering of the entire acceptor stem of *M. maezi* pyrrolysyl tRNA (tRNA^Pyl), leading to the identification of a highly active variant, PyOtR. PyOtR significantly improves the incorporation of various noncanonical amino acids (ncAAs) and facilitates the development of stable cell lines expressing ncAA incorporation machinery. The enhanced activity of PyOtR is demonstrated through improved ncAA incorporation efficiency, higher suppression efficiency of nonsense codons, and better performance in viral vector construction and stable cell line generation. These findings highlight the potential of VADER2.0 for expanding the capabilities of directed evolution in mammalian cells and advancing the technology of ncAA mutagenesis.