Owing to societal development and aging population, the impact of cancer on human health and quality of life has increased. Early detection and surgical treatment are the most effective approaches for most cancer patients. As the scope of conventional tumor resection is determined by auxiliary examination and surgeon experience, there is often insufficient recognition of tiny tumors. The ability to detect such tumors can be improved by using fluorescent tumor-specific probes for surgical navigation. This review mainly describes the design principles and mechanisms of activatable probes for the fluorescence imaging of tumors. This type of probe is nonfluorescent in normal tissue but exhibits obvious fluorescence emission upon encountering tumor-specific substrates, such as enzymes or bioactive molecules, or changes in the microenvironment, such as a low pH. In some cases, a single-factor response does not guarantee the effective fluorescence labeling of tumors. Therefore, two-factor-activatable fluorescence imaging probes that react with two specific factors in tumor cells have also been developed. Compared with single biomarker testing, the simultaneous monitoring of multiple biomarkers may provide additional insight into the role of these substances in cancer development and aid in improving the accuracy of early cancer diagnosis. Research and progress in this field can provide new methods for precision medicine and targeted therapy. The development of new approaches for early diagnosis and treatment can effectively improve the prognosis of cancer patients and help enhance their quality of life.