Normal physiological functions as well as regulatory mechanisms for various pathological conditions depend on the activity of enzymes. Thus, determining the in vivo activity of enzymes is crucial for monitoring the physiological metabolism and diagnosis of diseases. Traditional enzyme detection methods are inefficient for in vivo detection, which have different limitations, such as high cost, laborious, and inevitable invasive procedures, low spatio-temporal resolution, weak anti-interference ability, and restricted scope of application. Because of its non-destructive nature, ultra-environmental sensitivity, and high spatiotemporal resolution, fluorescence imaging technology has emerged as a potent tool for the real-time visualization of live cells, thereby imaging the motility of proteins and intracellular signalling networks in tissues and cells and evaluating the binding and attraction of molecules. In the last few years, significant advancements have been achieved in detecting and imaging enzymes in biological systems. In this regard, the high sensitivity and unparalleled spatiotemporal resolution of fluorescent probes in association with confocal microscopy have garnered significant interest. In this review, we focus on providing a concise summary of the latest developments in the design of fluorogenic probes used for monitoring disease-associated enzymes and their application in biological imaging. We anticipate that this study will attract considerable attention among researchers in the relevant field, encouraging them to pursue advances in the development and application of fluorescent probes for the real-time monitoring of enzyme activity in live cells and in vivo models while ensuring excellent biocompatibility.