Exploring and understanding the interaction of changes in the activities of various enzymes, such as proteases, phosphatases, and oxidoreductases with tumor invasion, proliferation, and metastasis is of great significance for early cancer diagnosis. To detect the activity of tumor-related enzymes, various molecular probes have been developed with different imaging methods, including optical imaging, photoacoustic imaging (PAI), magnetic resonance imaging, positron emission tomography, and so on. In this review, we first describe the biological functions of various enzymes and the selectively recognized chemical linkers or groups. Subsequently, we systematically summarize the design mechanism of imaging probes and different imaging methods. Finally, we explore the challenges and development prospects in the field of enzyme activity detection. This comprehensive review will provide more insight into the design and development of enzyme activated molecular probes.