Fluorescein is widely used in medical diagnostics, particularly in ophthalmology, to detect eye injuries or abnormalities. It is applied as a dye in fluorescein angiography to visualize blood flow in the retina and identify issues like retinal detachment or diabetic retinopathy. In addition, it is used in contact lens fitting to assess tear film dynamics and detect corneal abrasions or ulcers, often by applying a solution and observing fluorescence under blue light.

Beyond medical applications, fluorescein serves as a tracer in environmental studies to track water flow, study groundwater movement, and detect leaks in plumbing systems. Its fluorescent properties, activated by UV or blue light, also make it valuable in laboratory research for labeling and detecting biomolecules, such as in cell studies or blood flow in animal models.

Additionally, fluorescein is utilized in industry for producing fluorescent inks or dyes in printing and art, as well as in quality control processes requiring UV inspection.