An assay to detect carbon monoxide (CO), one of the gaseous signaling molecules, has been prepared using a new palladium complex probe. The ethylenediamine group linked to the naphthalimide fluorophore coordinates to Pd(II) which intramolecularly quenches the emission. Upon treatment with CO, the absorbance of the turn-on fluorescent sensor changes due to the formation of a complex between Pd(II) and CO at room temperature in a phosphate buffer. As the concentration of CO increases, the probe peak emission intensity at 527 nm gradually increases. Other analyte controls, such as K⁺, Mg²⁺, Al³⁺, Zn²⁺, Cr³⁺, Hg²⁺, Fe³⁺, alanine, glycine, leucine, lysine, serine, threonine, tyrosine, F^–, Cl^–, Br^–, NO, NO₂^–, NO₃^–, HCO₃^–, CH₃COO^–, H₂O₂, ^•OH, and ^tBuOO^•, exhibit no significant effect on emission intensity. The response time of the probe to CO was quite fast because of the relatively weak coordination of Pd(II) to the pendent ethylenediamine group. The Pd probe is capable of detecting CO in aqueous buffer as well as in living cells with high selectivity and stability, providing a potential real-time indicator for studying CO-involved reactions in biological systems.