A ratiometricfluorescent hybrid nanoprobe CDs-1 for arginine (Arg), exhibiting high sensitivity (the limit of detection, LOD, being 6.5 × 10-8 mol/L) and excellent selectivity and anti-interference ability, was fabricated through fluorescence resonance energy transfer (FRET) and the electrostatic attraction between positively-charged hemicyanine molecules and negatively-charged carbon dots (CDs). Arg can be quantitatively detected in the concentration range from 6.0 × 10-5 mol/L to 2.7 × 10-4 mol/L. Further, due to its ability to target mitochondrion and low cytotoxicity, intracellular Arg was successfully tracked through ratiometricfluorescence imaging.