A structurally simple, water-soluble rhodamine-derivatived fluorescent probe, which is responsive to acidic pH, was conveniently synthesized via a one-step condensation reaction of rhodamine B hydrazide and 4-formybenzene-1,3-disulfonate. As a stable and highly sensitive pH sensor, the probe displays an approximately 50-fold fluorescence enhancement over the pH range of 7.16-4.89 as the structure of probe changes from spirocyclic (weak fluorescent) to ring-open (strong fluorescent) with decreasing pH. The synthesized fluorescent probe is applied to the detection of pH changes in vitro and in vivo bioimaging of immortalized gastric cancer cells, with satisfactory results.