Carbon monoxide (CO) is one of the crucial gasotransmitters in biological systems. However, the exploration of its biological and pathological roles is hindered by the limited availability of functional molecular tools. In this study, a new aldol-propargyl incorporated fluorescent probe (HAP) has been developed to detect CO. Upon reaction with CO and Pd2+, the probe exhibits a significant increase in emission intensity at 596 nm. The detection process involves the Pd-catalyzed depropargylation reaction, resulting in the formation of a highly fluorescent product. Furthermore, the probe toward CO in the presence of Pd2+ displays several advantageous features, including a low detection limit of 0.35 μM, a large Stokes shift (256 nm), and a rapid response time of just 50.0 s. It also displays excellent selectivity toward CO, remaining unresponsive to other interfering species. The probe HAP was successfully applied for the quantitative detection of CO in environmental soil samples utilizing a smartphone. Additionally, the probe HAP was incorporated into test strips, enabling portable and sensitive detection of CO under UV light. Furthermore, low cytotoxic HAP was employed to image CO in HeLa cells. Therefore, this work presents a promising platform for ultra-fast, portable, smart, and multifunctional monitoring of CO, contributing to human health and ecological protection efforts.