We developed a novel label-free strategy for electrochemical detection thrombin based on rolling circle amplification (RCA) triggering poly adenine production for adsorption gold nanoparticles (AuNPs). In this assay, the aptamer 1 was first immobilized on the gold electrode (GE) surface as the capture probe via poly adenine-Au. Subsequently, the thrombin and the aptamer 2 were deposited on the electrode surface. The aptamer 2 fragment on the sensor surface as a primer hybridized with the RCA template to initiate the RCA process, which generated massive long DNA sequences that contained many adenines. Subsequently, the AuNPs was absorbed on the long-repeated adenines of RCA product, resulting in the multiplication of AuNPs on the electrode surface, which were used for subsequent electrocatalytic reduction of H2O2. In this sensing system, the electrochemical signals with the concentration of thrombin over a range from 0.1 pM to 10 nM were detected and the detection limit obtained was 35 fM, and have high selectivity toward its target protein. In addition, due to the high affinity between poly adenine and GE or AuNPs, the detection can be carried out without any fussy modification process, thus had a promising application in clinical diagnosis.