A foundation of genetic code expansion is the evolution of orthogonal aminoacyl-tRNA synthetase to recognize a non-canonical amino acid, which typically involves read-through of amber stop codon in the genes used in selection. The process is time-consuming, labor-intensive, and susceptible to certain bias. As a complementation, herein, we rationally designed a function-based method to directed evolution of aminoacyl-tRNA synthetase for acylated lysine derivatives.