Abstract A novel fluorescent probe for extreme acidity is developed using biocompatible indole derivative as the fluorophore. The probe is linearly proportional to extreme acidity range of 2.3–3.6 with pKa 2.89. Moreover, it displays promising spectroscopic features such as large Stokes shift (145 nm), highly selective response to H+ over various metal ions, good photostability and excellent reversibility. The confocal laser scanning microscope images display the probe possesses excellent cell membrane permeability, and is further applied successfully to monitor pH fluctuations of extreme acidity range in E. coli cells without influence of autofluorescence and native cellular species in biological systems.