A new fluorescent probe 1, bearing a pyridine group as receptor for H+and a coumarin dye as fluorophore, was synthesized and characterized by1H NMR,13C NMR, ESI-Ms and single crystal X-ray diffraction analysis. The probe exhibited fluorescence ratiometric response to acidic pH. With decreasing of the pH from 8.32 to 2.49, the fluorescence emission spectra exhibited large red shift from 541 to 631 nm, with the emission ratios (I541/I631) changed dramatically from 25.9 to 0.08, and the pKavalue was calculated to be 5.45. Probe 1 exhibited high selectivity to pH, other interference species including metal ions and amino acid exerted no visible effect on probe 1 detecting pH. The intracellular pH imaging applications proved that the probe is suitable for monitoring acidic pH fluctuations in living cells.