Herein, we report a novel signal-amplifiable self-assembling fluorescent nanoprobe for turn-on detection of specific enzyme activity. This probe employed a substrate of matrix metalloproteinase 2 (MMP2) together with a self-assembling unit, and showed a detection limit of less than 830 pM, which is 84-fold lower than that of our previous ligand-tethered fluorescent nanoprobes. Using this new probe, we succeeded in monitoring the specific activity of secreted MMP even in the conditioned media of tumor cells.