A highly sensitive electrochemical aptasensor is developed for the quantification of human a-thrombin (TB), where the response signal is amplified through the recycling of a functional DNA architecture (A1-TB-A2) driven by hairpin strand displacement. This DNA architecture is formed by the proximal binding of TB with two affinity strands (A1 and A2) encoding specific TB aptamer. Only in this case can the unpaired segments cooperatively hybridize with a recognition strand (RS) confined in a Watson-Crick and Hoogsteen triplex-helix (TH), along with the releasing of a DNA hairpin 1 (H1). Upon addition of another hairpin 2 (H2), the complementary base pairing of RS and H2 accelerates the repeated liberation and recycling of A1-TB-A2, bringing H1 releasing and switching into hairpin conformation. As such, the folded H1 with negative charges electrostatically attracts redox mediator [Ru(NH3)(6)](3+) to produce significantly enhanced current signal. After incubated with Nt.BstNBI, the folded H1 is cleaved at Nt.BstNBI-recognizable nicking site, resulting in decrease of electrostatic attraction to [Ru(NH3)(6)](3+). The electrochemical response dependent on TB is greatly amplified. Via rational integration of a protein-responsive DNA architecture recycling and enzyme-nicked amplification, this electrochemical strategy is highly specific and sensitive to TB with a limit of detection down to 32 fM, which would be potential to monitor diverse biomolecules. (C) 2019 The Electrochemical Society.