Protein engineering is an emerging field for developing novel therapeutic proteins and commercial enzymes, along with a major impact on the global market. In recent decades, advanced methods employing protein modification through expansion of the genetic code have led to the development of proteins with new biochemical and physical properties. These techniques have produced engineered proteins with improved attribute comprising substrate relaxation, protein drug conjugation and high stability under extreme conditions of high temperatures, pH and organic solvents. Furthermore, residue specific incorporation is the simplest method for the global incorporation of non-canonical amino acid (NCAA) for protein modification; however it has the major drawbacks of high production cost and manpower requirement. In the present study, we developed a method for the incorporation of single NCAA in two different proteins by using Escherichia coli (E. coli) expression system. For that, the dual protein expressing Escherichia coil JW2581 strain was constructed by transforming pQE8OL and pD881-PpiBT vectors with different promoters, selectable markers and AnnexinV, GFPHS gene. To modify the protein, the 3,4 dihydroxy phenyl alanine (DOPA) was globally incorporated into the GFPHS and Annexin V protein using dual protein expression system. The incorporation efficiency during the dual protein expression was achieved through optimized concentrations of amino acids, carbohydrate and inducers in minimal medium. This method for the incorporation of single NCAA into two different proteins using a single expression host system saves the production cost, manpower and time substantially.