Mitochondrial membrane potential (ΔΨm) is an important biophysical parameter playing central roles in cell apoptosis, mitochondrial dysfunction, and other biological and pathological processes. Herein, we have rationally designed and fabricated a unique fluorescent probe for convenient ΔΨm visualization based on hot-band absorption and controllable anti-Stokes shift emission. The robust probe was excitable via hot-band absorption and emitted anti-Stokes upconversion emission and Stokes downconversion fluorescence simultaneously. The anti-Stokes emission could be efficiently inhibited upon the binding to RNA. The cationic probe targeted mitochondria in living cells with high ΔΨm and displayed both anti-Stokes green emission and ordinary red fluorescence. After the decrease of ΔΨm, the probe immigrated out of mitochondria to RNA and nucleolus, which showed only red emission owing to the inhibition of anti-Stokes fluorescence. In this manner, the ΔΨm could be visualized in dual-color mode. The probe enabled clearly monitoring the reversible changes in ΔΨm and was successfully employed to visualize oxidative damage of living cells. The decrease of ΔΨm in living tissues was also successfully observed with the newly designed probe.