As an essential gasotransmitter, carbon monoxide (CO) had gradually become a research hotspot in that it possessed important physiological functions and unique pharmacological properties. However, to date, no report has focused on the topic of detecting CO both in vivo and using films. To open up a new field of CO probes, for the first time, we designed a probe (PMAH-CO) that showed a distinctive ratio emission characteristic and displayed the quantitative distribution of CO in HeLa cells and zebrafish with a higher signal-to-noise ratio. Meanwhile, the fluorescent polysiloxane-based film (PMF) containing PMAH-CO exhibited an excellent response to CO. Due to the addition of the Si-O bond, the probe exhibited a broad transparency in the visible light range and had excellent photostability. Moreover, the probe was economically viable, easy to handle, and suitable for biological research. Hence, PMAH-CO and PMF would open up the road to broaden the application of silicone materials in the field of fluorescence imaging.