We herein describe a novel method for the detection of target protein based on the target-responsive DNA polymerase activity. In the sensor, two different types of DNA aptamers with the respective functions: one binds to the target protein and the other binds to DNA polymerase, are rationally engineered and combined to form the detection probe that regulates DNA polymerase activity in response to the target protein. In the presence of target protein, the detection probe becomes destabilized and stops the inhibition of DNA polymerase activity. Consequently, the active DNA polymerase initiates the primer extension reaction on the target-specific DNA aptamer, which recycles the target protein to promote another activation cycle of DNA polymerase. In addition, DNA polymerase also catalyzes the primer extension reaction on the primer/template complex in conjugation with TaqMan probe, leading to the significantly enhanced fluorescence intensities. With this novel strategy, we detected a model target protein, lysozyme with a limit of detection as low as 0.80 nM. In addition, the practical applicability of this system was successfully demonstrated by determining lysozyme in human serum. (C) 2019 Elsevier B.V. All rights reserved.