Proteases mediate a wide variety of biological events and have a critical role in the development of many diseases. Protease detection methods can be hindered by the limitation of assay safety, sensitivity, specificity, time constraints and ease of on-site analysis. Notably, the implementation of various detection methods on biosensing platforms translates them into practical biosensing applications. Currently, the detection of prostate cancer and AIDS at the earliest occasion is one of the major research obstacles. Therefore, recent advances focus on the development of portable detection systems toward point-of-care testing. These detection systems should be highly sensitive and specific for the detection of their prognostic biomarkers, such as the prostate-specific antigen and HIV load assay for prostate cancer and AIDS, respectively. These methods will also facilitate decision-making on a treatment regimen.