L-Dihydroxyphenylalanine (L-DOPA) is a naturally occurring amino acid with interesting biochemical properties. The amino acid has been used for the treatment of Parkinson’s disease and various biochemical applications. In this report, L-DOPA was biosynthesized from a tyrosine phenol-lyase (TPL) by using catechol, pyruvate, and ammonia, as starting materials, and the biosynthesized amino acid directly incorporated into proteins by an evolved aminoacyl-tRNA and aminoacyl-tRNA synthetase (aaRS) pair. The aaRS used for the L-DOPA incorporation was selected from an aaRS library, and showed better efficiency than the previously reported aaRS. This direct incorporation system showed efficient L-DOPA incorporation with no incorporation of Tyr, and better protein yield than the conventional incorporation system using L-DOPA. Therefore, by using the biosynthetic system, mutant proteins containing L-DOPA could be produced with less expanse and better yield. This approach could be useful for a large scale protein production for pharmaceutical and industrial application, and provide an impetus for expansion of biosynthesis of unnatural amino acids to more challenging and interesting amino acids.