4-Cyanotryptophan is a blue fluorescent unnatural amino acid (UAA) that not only has a large fluorescence quantum yield (>
0.8) and a long fluorescence lifetime (>
13 ns) in water but is also chemically and photophysically stable, making it a valuable UAA-based fluorophore in biological spectroscopy and microscopy. However, its broad utility requires further development of simple methods for introducing this UAA into large proteins. Herein, we demonstrated two such methods, one is based on a post-translational modification strategy and the other is based on a selective pressure incorporation approach. In addition, in a proof-of-principle application, we demonstrated that incorporation of this UAA at the 66 position of a green fluorescent protein (GFP) variant can modify its photophysical properties.