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Copper is one of the most essential transition metal in human body which serves as an essential cofactor for oxidative scavenging enzymes. Due to its redox property it has the ability to generate reactive oxygen species and play an important role in metabolism. Imbalance of copper homeostasis leads to diseases such as Menkes, Wilson’s disease and also in Alzheimer’s disease. So, it would be highly advantageous if we develop an efficient method for measuring copper through spectroscopic /fluoro spectroscopic methods without addition of any reagents. As we know that GFP is already used as fluorescent tag in prokaryotic system as well as in mammalian systems, so developing GFP based in vivo copper biosensing tag will help in identifying the copper based diseases and localization of copper ions in cell. In the current study we have developed a metal biosensor through unnatural amino acid mutagenesis where the metal chelating amino acid, L-DOPA was metabolically incorporated into GFP. The fluorescence of DOPA incorporated GFP specifically quenches in the presence of copper and fluorescence of the protein was recovered after adding a metal chelator. This strategy helps to develop a fluorescent protein based copper biosensor and also helps in developing microarray based copper quantitation