A fluorescence ratiometric probe 1 for cysteine (Cys) and homocysteine (Hcy) has been rationally constructed based on intramolecular charge transfer (ICT) mechanism. Upon treatment with Cys/Hcy, probe 1 exhibited a fluorescence ratiometric response, with the emission wavelength displaying a large shift (from 526 nm to 446 nm). When 90 μM Cys were added, the emission ratios (I₄₄₆/I₅₂₆) of the probe changed dramatically from 0.01797 to 4.65472. The detection limit was also measured to be 0.18 μM (S/N = 3). The theoretical calculations have confirmed that the ratiometric response of probe 1 to Cys/Hcy is due to the inhibition of ICT process upon the reaction of probe 1 with Cys/Hcy. Furthermore, the fluorescence imaging experiments in living cell demonstrated that probe 1 was favourable for intracellular Cys/Hcy imaging.