Proteases, enzymes that hydrolyze peptide bonds, have various applications in medicine, clinical applications, and pharmaceutical development. They are used in cancer treatment, wound debridement, contact lens cleaning, prion degradation, biofilm removal, and fibrinolytic agents. Proteases are also crucial in cardiovascular disease treatment, emphasizing the need for safe, affordable, and effective fibrinolytic drugs. Proteolytic enzymes and protease biosensors are increasingly used in diagnostic and therapeutic applications. Advanced technologies, such as nanomaterials-based sensors, are being developed to enhance the sensitivity, specificity, and versatility of protease biosensors. These biosensors are becoming effective tools for disease detection due to their precision and rapidity. They can detect extracellular and intracellular proteases, as well as fluorescence-based methods for real-time and label-free detection of virus-related proteases. The active utilization of proteolytic enzymatic biosensors is expected to expand significantly in biomedical research,