Nile Blue A (Nile blue sulfate) is employed for differentiating melanins and lipofuscins, staining fats, and preparing amperometric glucose sensors.

Nile blue A is an effective stain for PHB (polyhydroxybutyrate) granules in bacteria, outperforming Sudan black B in this role. It produces a strong orange fluorescence in poly-p(3-hydroxybutyrate) granules and stains a greater number of PHB granules than Sudan black B, while also being less susceptible to being washed out of the cell during decolorization procedures. Aside from staining polyhydroxyalkanoic acid-accumulating microorganisms or detecting polyhydroxyalkanoic acids in them, Nile blue A can stain Escherichia coli cells lacking detectable polyhydroxyalkanoic acids without interfering with the surface display or specific labeling of peptides. It offers a convenient and cost-effective method for staining E. coli in flow cytometry, serving as an alternative to other fluorescent dyes or intracellular expression markers like green fluorescent protein. Additionally, Nile blue A acts as a powerful photosensitizer in photodynamic therapy, spreading through the body via the bloodstream upon intravenous administration and interacting with various biomolecules in most cells.