We prepared a water-soluble conjugated polymer composed of electron-donating units and electron-accepting groups in the backbone. The polymer exhibits a short wavelength (blue) emission in aqueous solution and long wavelength (red) emission in the solid state, because of intermolecular energy transfer. Considering this, we develop a new approach for the sensitive detection of trypsin, which is known to control pancreatic exocrine function, using an ensemble system composed of the anionically charged conjugated polymer and cationically charged polypeptides (such as polylysine and polyarginine). The blue-emitting, water-soluble conjugated polymer becomes aggregated upon exposure to the polypeptides, leading to a red-emitting assay ensemble. The red-emitting assay ensemble becomes dissociated in the conjugated polymer and polypeptide fragments by selective degradation of trypsin, which then exhibits recovery of blue emission. This emission-tuning assay ensemble allows for detection of trypsin at nanomolar concentrations, which enables naked-eye detection. Importantly, this strategy can be employed for label-free, continuous assay for trypsin.