Chromophore-assisted light inactivation (CALI) is a significant technique to realize knockdown or loss-of-function of target protein molecules in a spatiotemporally regulated manner. Based on the advantages of CALI, we hybridized PPET3-N2, a well-studied cation conjugated polyelectrolyte (CP), with a label-free DNA containing aptamer via electronic and hydrophobic interactions to produce reactive oxygen species (ROS) under light irradiation, which resulted in specific inactivation of the protein of interest, thrombin. The aptamer introduced into the system formed a complex with thrombin, whereby the aptamer decreased the distance between the PPET3-N2/DNA hybrid and thrombin. This proposed strategy efficiently inactivated thrombin with good specificity and provides a novel and universal approach for controlled inactivation of biomolecules.