Fluorescein is a fluorophore commonly used in microscopy. It is also used in the gain medium in a type of dye laser. Also used in forensics to detect latent blood stains serology.

In biology the isothiocyanate derivative of fluorescein is often used to label and track cells in fluorescence microscopy applications. The fluorescence of this molecule is very high, and excitation occurs at 494nm and emission at 525nm. The isothiocyanate group (-N=C=S) replaces a hydrogen atom on the bottom ring of the structure, and is reactive with amine groups on proteins inside cells. Fluorescein isothiocyanate is often abbreviated as FITC. Additional biologically active molecules (such as antibodies) may also be attached to fluorescein, allowing biologists to target the fluorophore to specific proteins or structures within cells.

One of its more recognizable uses is in the Chicago River. Every St. Patrick's Day, fluorescein is used to dye the river green.