Sensitive and facile detection of biomarkers is essential for early diagnosis and treatment of diseases. To this end, we here proposed a colorimetric protease assay by the modular combination of proteolysis-responsive transcription and spherical nucleic acids (SNAs). In this assay, target protease-mediated proteolysis triggers the synthesis of RNAs by in vitro transcription, which subsequently results in the aggregation of SNAs with remarkable redshifts in the wavelength of surface plasmon resonance-related absorption. As a proof of concept, this assay achieved the sensitive and specific detection of matrix metalloprotease-2 (MMP-2) with a limit of detection of 3.3 pM. Moreover, the applicability of this colorimetric assay can be expanded to other protease biomarkers (e.g., thrombin and hepatitis C virus NS3/4A) by tuning the target-responsive RNA polymerase module. Furthermore, by the immobilization of SNAs on a glass fiber membrane, a test strip that enables the portable detection of target protease with a smartphone was developed. With the use of a mobile application to capture and process the colorimetric signals, this portable detection system allowed for sensitive evaluation of MMP-2 levels in biological and clinical specimens, highlighting its potential in point-of-care diagnosis of diseases.