The preparation of an electroactive matrix for the immobilization of the bioprobe shows great promise to construct the label-free biosensors. Herein, the electroactive metal-organic coordination polymer has been in-situ prepared by pre-assembly of a layer of trithiocynate (TCY) on a gold electrode (AuE) through Au-S bond, followed by repetitive soaking in Cu(NO3)2solution and TCY solutions. Then the gold nanoparticles (AuNPs) and the thiolated thrombin aptamers were successively assembled on the electrode surface, and thus the electrochemical electroactive aptasensing layer for thrombin was achieved. The preparation process of the biosensor was characterized by an atomic force microscope (AFM), attenuated total reflection-Fourier transform infrared (ATR-FTIR), and electrochemical methods. Electrochemical sensing assays showed that the formation of the aptamer-thrombin complex changed the microenvironment and the electro-conductivity of the electrode interface, causing the electrochemical signal suppression of the TCY-Cu2+polymer. Additionally, the target thrombin can be label-free analyzed. Under optimal conditions, the aptasensor can detect thrombin in the concentration range from 1.0 fM to 1.0 μM, with a detection limit of 0.26 fM. The spiked recovery assay showed that the recovery of the thrombin in human serum samples was 97.2–103%, showing that the biosensor is feasible for biomolecule analysis in a complex sample.