Nowadays, ozone has been widely applied in industry and medical therapies. However, excessive exposure to ozone can lead to lung dysfunction and many respiratory symptoms. As a member of reactive oxygen species (ROS), ozone was also involved in various physiology and pathology process. Given the fact of this, the effective detection of ozone in the atmosphere and biological system is of vital significance. Herein, we reported a novel dicyanomethylene-4H-pyran (DCM)-based fluorescent probe DCM-O3 with butenyl being the recognition moiety for monitoring ozone. The probe displayed high selectivity towards ozone, and its response towards ozone could be completed within 5 min under the optimal condition. Besides, a good linear correlation was obtained between the ozone concentrations (0–50 μM) and the corresponding fluorescent intensity at 560 nm, and the limit of detection (LOD) was calculated to be 6.2 × 10−7 M. Moreover, the probe DCM-O3 showed low cytotoxicity and was successfully applied to detect ozone in live cells. Given all the merits, the probe DCM-O3 could function as a robust tool for researchers to investigate ozone-related diseases in the complex biological environment.