Recently, L-4-cyanotryptophan has been shown to be an efficient blue fluorescence emitter, with the potential to enable novel applications in biological spectroscopy and microscopy. However, lack of facile synthetic routes to this unnatural amino acid limits its wide use. Herein, we describe an expedient approach to synthesize Fmoc protected L-4-cyanotryptophan with high optical purity (>99%). Additionally, we test the utility of this blue fluorophore in imaging cell-membrane-bound peptides and in determining peptide–membrane binding constants.