Signal amplification is an effective way to achieve sensitive analysis of biomarkers, exhibiting great promise in biomedical research and clinical diagnosis. Inspired by the transcription process, here we present a versatile strategy that enables effective amplification of proteolysis into nucleic acid signal outputs in a homogeneous system. In this strategy, a protease-activatable T7 RNA polymerase is engineered as the signal amplifier and achieves 3 orders of magnitude amplification in signal gain. The versatility of this strategy has been demonstrated by the development of sensitive and selective assays for protease biomarkers, such as matrix metalloproteinase-2 (MMP-2) and thrombin, with sub-picomole sensitivity, which is 4.3 × 103-fold lower than that of the standard peptide-based method. Moreover, the proposed assay has been further applied in the detection of MMP-2 secreted by cancer cells, as well as in the assessment of MMP-2 levels in osteosarcoma tissue samples, providing a general approach for the monitoring of protease biomarkers in clinical diagnosis.