Sulforhodamine B is widely used as a fluorescent dye in biological and medical research. It is commonly employed in cell viability and cytotoxicity assays due to its ability to bind to cellular proteins under acidic conditions, allowing quantification of adherent cells. The dye is also used in high-throughput screening for anticancer drug development, where it helps measure the effect of compounds on cell growth and survival.

In addition, Sulforhodamine B serves as a tracer in environmental studies, such as tracking water flow and studying sediment transport, thanks to its strong fluorescence and stability in aqueous environments. Its bright red fluorescence makes it suitable for use in fluorescence microscopy and flow cytometry applications when appropriate filters are used.

The compound is also utilized in laser technology as a gain medium in dye lasers, producing visible light in the red to near-infrared range. Due to its photostability and high quantum yield, it is preferred in imaging and photophysical experiments where consistent fluorescence output is required.