Fluorescence spectroscopy is a powerful tool for probing complex biological processes. The ubiquity of peptide–protein and protein–protein interactions in these processes has made them important targets for fluorescence labeling, and to allow sensitive readout of information concerning location, interactions with other biomolecules, and macromolecular dynamics. This review describes recent advances in design, properties and applications in the area of fluorescent amino acids (FlAAs). The ability to site-selectively incorporate fluorescent amino acid building blocks into a protein or peptide of interest provides the advantage of closely retaining native function and appearance. The development of an array of fluorescent amino acids with a variety of properties, such as environment sensitivity, chelation-enhanced fluorescence, and profluorescence, has allowed researchers to gain insights into biological processes, including protein conformational changes, binding events, enzyme activities, and protein trafficking and localization.