5(6)-Nitrofluorescein is widely used as a fluorescent dye in various biological and chemical applications. Its primary use is in fluorescence microscopy, where it serves as a marker to visualize and track cellular components or processes. The compound is also employed in assays to detect and quantify enzymatic activities, particularly those involving hydrolases or peroxidases, due to its ability to produce a strong fluorescent signal upon reaction. Additionally, it is utilized in pH-sensitive studies, as its fluorescence intensity changes with variations in pH, making it a useful probe for monitoring pH changes in biological systems. In research, it is often incorporated into sensor designs for detecting specific analytes or environmental conditions. Its versatility and strong fluorescence properties make it a valuable tool in both academic and industrial laboratories.