Mitochondria are iron-rich organelles that are involved in the process of energy production through the electron-transporting system and heme synthesis. We developed a new mitochondria-targeted fluorescent probe, MtFluNox/Ac-MtFluNox, for Fe(ii) based on N-oxide chemistry, which we recently established as a Fe(ii)-selective fluorogenic switch. The deacetylated form MtFluNox showed a turn-on response towards Fe(ii) with high metal selectivity in cuvette experiments, and an imaging study using its cell-compatible analogue Ac-MtFluNox demonstrated mitochondria-specific fluorescence enhancement in response to Fe(ii) in living cells. Furthermore, the probe was able to detect endogenously accumulated Fe(ii) induced as a result of the inhibition of heme synthesis.