Because of the biological importance of CO, the development of effective probes for the detection of CO in living systems is of great significance. In particular, near-infrared (NIR) fluorescence probes with good water solubility and large Stokes shift are indispensable tools for CO detection in vivo. However, such tools are extremely scarce. Herein, a modified rhodol dye was used to develop a novel NIR fluorescent probe (Rh-NIR-CO) for selective and sensitive detection of CO using the Pd0-mediated Tsuji-Trost reaction. This probe shows good water solubility and rapid CO detecting ability in aqueous buffer at pH 7.4, accompanied by distinct colorimetric and turn-on NIR emission changes at 676 nm with a large Stokes shift (135 nm) and low detection limit (37 nM). Moreover, NIR fluorescence imaging of CO in living cells, zebrafish, and living mice was successfully applied with this probe. These excellent results highlighted Rh-NIR-CO as a promising new tool for in vitro and in vivo detection of CO.