Abstract A fluorescein‐based fluorescent probe has been designed and synthesised that selectively detects H 2 S in aqueous medium, among various analytes tested. This fluorescein‐based fluorescent probe has also been successfully utilised for real‐time imaging of exo‐ and endogenously produced H 2 S in cancer cells and normal cells. Moreover, the probe can also detect H 2 S in the rat brain hippocampus at variable depths and in living nematodes.