A regenerable electrochemiluminescence aptasensor for thrombin (TB) has been developed using dual co-reactants of poly(ethylenimine) (PEI) and thiosemicarbazide (TSC) for signal amplification. Herein, Au nanoparticle (AuNPs) were in-situ electrodeposited on glassy carbon electrode (GCE) in advance, and then the colloidal gold nanoparticle (nano-Au) and TSC were alternately self-assembled on AuNPs/GCE to obtain nano-Au/TSC/AuNPs multi-layers for 5'-thiolated partly complementary strand (PCS) immobilization. Subsequently, the multifunctional binding aptamer (MBA), consisted of an adenosine triphosphate (ATP) binding aptamer (ABA) and a thrombin binding aptamer (TBA), was hybridized with PCS to generate double-strands DNA (PCS/MBA). Meanwhile, the signal probe was fabricated by immobilizing detection thrombin aptamer (TBA 2) on the surface of PEI enhanced Ru(bpy)(3)(2+) doped nanospheres. On the basis of sandwich-type tactics, TB was measured quantitatively in the range from 1 x 10(-5) nM to 0.1 nM. More importantly, the aptasensor could regenerate by treating with ATP to take off MBA based on ATP-induced conformation change, leaving only PCS onto the modified electrode to rehybridize with MBA. The proposed aptasensor exhibited good selectivity, acceptable stability and satisfactory regenerability. (C) 2015 Elsevier Ltd. All rights reserved.