GSH, Cys and Hcy are the main intracellular thiols to play crucial roles in human pathologies. It is a great challenge to differentiate these three biothiols using single molecular fluorescent probes due to their close similarities in chemical structure and reactivity. In this work, based on the fluorescence resonance energy transfer (FRET) mechanism, a fluorescent probe CPR was constructed to simultaneously distinguish GSH and Cys/Hcy by means of ratiometric fluorescence changes: from red (584 nm) to green (542 nm) for GSH and from red (584 nm) to blue (472 nm) for Cys/Hcy. This probe showed high sensitivity and selectivity with low limits of detection (LOD = 12 nm, 13 nm and 30 nm for Cys, Hcy and GSH, respectively) and was capable of imaging GSH and Cys/Hcy in cells and zebrafish in a ratiometric manner with low toxicity.