Tumor-associated proteases (TAPs), such as legumain, are actively involved in cancer progression; they have been used as biomarkers for diagnosis, prognosis, and drug targeting. As a result, in-vivo detection and trafficking of TAPs have attracted a great deal of attention. TAP-specific probes for in-vivo imaging, however, remain rare. A TAP-responsive hybrid nanoprobe system based on quantum dots (QD) and the fluorescence resonance energy transfer (FRET) effect is presented for the detection of legumain (asparaginyl endopeptidase), which is overexpressed in many tumors. A novel hybrid construction method is developed for fabricating the nanoprobe, by which the strong heparin-protamine affinity is used for conjugation. The hybrid comprises two components: 1) low-molecular-weight heparin (LH)-modified QD, and 2) low-molecular-weight protamine (LMWP)-conjugated fluorescence quencher QSY21, through a legumain-cleavable linker. The hybrid nanoprobe (i.e., a FRET system) is self-assembled via the LH-LMWP affinity. The linker between LMWP and QSY21 is selectively cleaved by legumain, leading to QSY21 detachment and fluorescence recovery in the tumor. In-vivo imaging is successfully achieved in the colon tumor mouse model. Importantly, such a hybrid nanoprobe system is adaptable for the detection of other TAPs (e. g., matrix metalloproteinase -2) by using an established, corresponding substrate-peptide linker, thereby offering a universal platform for TAP detection and tumor imaging.