In this work, a new signal amplified strategy based on the quenching effect of hemin and Au nanoparticles decorated CeO 2 nanoparticles (Au@CeO 2 NPs) for ultrasensitive detection of thrombin (TB) is reported for the first time. Herein, the poly(ethylenimine) (PEI) enhanced Ru(bpy) 3 2+ nanocomposite was implemented by direct chemical polymerization, which could provide the desirable enhanced initial ECL signal. Furthermore, the detection aptamer of thrombin (TBA 2) was immobilized on Au@CeO 2 NPs to form TBA 2/Au@CeO 2 conjugates. Then, the G-rich DNA of TBA 2 sequence could fold into a G-quadruplex structure to embed hemin to obtain the quenching probe of hemin/TBA 2/Au@CeO 2 conjugates. In the presence of target TB, the sandwiched structure could be formed between capture aptamer (TBA 1), TB and hemin/TBA 2/Au@CeO 2 conjugates, thereby resulting in a proportional quenching in ECL response with TB, due to the quenching of both hemin and Au@CeO 2 NPs. As a result, the signal-off aptasensor showed a wider linear range response from 10 −13 to 10 −8 M with lower detection limit of 0.03pM.