Eosin Y is the most common counterstain to alum hematoxylin in the Hematoxylin and Eosin method (H&E). It stains satisfactorily from both aqueous and ethanolic solution. It is one of the dyes in Papanicolaou's EA solutions for staining exfoliative cytology for cervical cancer screening. It can be used to make Romanowsky stains. It is widely used in numerous procedures. It is strongly fluorescent, but this property is hardly ever used.

The free radical, eosinol Y is made by treating the dye with hydrochloric acid. It is sometimes called eosin Y, spirit soluble. Eosinol Y is soluble in ethanol and slightly soluble in xylene, but not in water, and is useful for staining difficult tissues.