AbstractThe exploration of advanced probes for cancer diagnosis and treatment is of high importance in fundamental research and clinical practice. In comparison with the traditional “always‐on” probes, the emerging activatable probes enjoy advantages in promoted accuracy for tumor theranostics by specifically releasing or activating fluorophores at the targeting sites. The main designing principle for these probes is to incorporate responsive groups that can specifically react with the biomarkers (e. g., enzymes) involved in tumorigenesis and progression, realizing the controlled activation in tumors. In this review, we summarize the latest advances in the molecular design and biomedical application of enzyme‐responsive organic fluorescent probes. Particularly, the fluorophores can be endowed with ability of generating reactive oxygen species (ROS) to afford the photosensitizers, highlighting the potential of these probes in simultaneous tumor imaging and therapy with rational design. We hope that this review could inspire more research interests in the development of tumor‐targeting theranostic probes for advanced biological studies.