A novel acidic fluorescent probe 1 has been designed, synthesized, characterized and evaluated in vivo as optical imaging of intracellular H(+). The design strategy for the probe is based on the change in structure between spirocyclic (non-fluorescent) and ring-open (fluorescent) forms of rhodamine dyes. The probe exhibits high sensitivity, good photostability, excellent cell membrane permeability and strong pH dependence. The pH titration indicates that the fluorescence intensity increases more than 100-fold within the pH range of 4.2-6.0 with the pK(a) value of 4.85, which is valuable for studying acidic organelles in living cells. The fluorescent imaging of HepG2 cells also demonstrates that the designed probe has great value in monitoring intracellular H(+) within living cells.