Carbon monoxide (CO) has attracted considerable attention because of its therapeutic potential and its role in cellular signalling and biological systems. However, CO is highly toxic even at low concentrations and is difficult to detect because of its colourless and odourless nature. Therefore, the rapid, sensitive, and in situ monitoring of the levels of CO in living cells is essential for understanding its biological functions and preventing toxic effects. Herein, a novel fluorescent “off–on” probe was developed for the recognition of CO based on a Pd⁰-mediated Tsuji–Trost reaction. The probe showed good sensitivity (0.14 μM), fast response (~8.0 min) and excellent selectivity toward CO. The sensing mechanism was verified by FTIR, TOF-MS, and 1H NMR analyses. Additionally, a smartphone-assisted platform based on the probe allows quick, on-site quantitative analysis of CO in solution phase and portable test strips without relying on sophisticated instruments, demonstrating its potential as a smartphone-assisted fluorescence sensing platform for CO monitoring. Furthermore, the developed sensing platform not only allows accurate determination of CORM-2-derived CO in spiked soil samples but also enables fluorescence imaging of CO in HeLa cells, demonstrating its applicability in biological systems. Overall, this study provides a robust and versatile probe for CO monitoring with significant potential for environmental analysis and biological applications.