Carbon monoxide (CO) not only causes damage to life and health as an environmental pollutant, but also undertakes many physiological functions in organisms. In particular, developing means that can be used for the determination of CO in organelles will provide insight into the vital role it plays. Studies have shown that mitochondrial respiration is closely related to CO concentrations, so it is critical to develop tools for CO detection in mitochondria. Here, we use a rhodamine derivative that can target mitochondria as fluorophores to construct a mitochondrial-labeled CO fluorescence probe (Rh-CO) with high sensitivity (detection limit: 9.4 nM), excellent water-solubility, and long emission (λem = 630 nm). Prominently, the probe has outstanding mitochondria-targeting capabilities. Moreover, we used transient glucose deprivation (TGD) and heme to stimulate endogenous CO production in living cells and zebrafish, respectively, and the probe exhibited excellent imaging capabilities. All in all, we expect this probe to contribute to a deeper understanding of the role played by CO in mitochondria.