The present invention relates to a method of producing a phosphorylated protein using a SepRS (O-phosphoseryl-tRNA synthetase) mutant and an EF-Tu mutant, which have increased activity. More specifically, the invention relates to a method of producing a phosphorylated protein by incorporating phosphoserine into the specific position of a target protein or polypeptide using tRNASep serving to recognize at least one codon in the mRNA of the target protein or polypeptide, an O-phosphoseryl-tRNA synthetase (SepRS) mutant selected by a molecular evolution technique and serving to aminoacylate tRNASep with phosphoserine (Sep), and an EF-Tu mutant serving to bind and deliver Sep-tRNASep to the ribosome.According to the invention, a phosphorylated protein can be produced in an amount of mg per liter using the SepRS and EF-Tu mutants. Thus, the invention is useful for the production of various phosphorylated proteins, including phosphorylated enzymes, the production of antibodies, the fabrication of protein chips, and cell-based screening for new drug development.