A novel protocol of electrochemical thrombin aptasensor based on a goldnanoparticles/thionine-graphene (AuNPs/Th-G) nanocomposite modified glassy carbon electrode was presented. Graphene was non-covalently functionalized by thionine viaπ-stacking interaction, and then AuNPs were electrodeposited onto the Th-G surface. The morphology and conductivity of the AuNPs/Th-G nanocomposite were characterized by scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The ethanethiol-substituted oligonucleotide probe was immobilized onto the surface of the AuNPs/Th-G nanocomposite to prepare an electrochemical biosensing platform. The aptamer-thrombin reaction was monitored by differential pulse voltammetry. Under optimum conditions, the proposed biosensor exhibits high sensitivity and a low detection limit for thrombin determination. The thrombin could be quantified in a wide range of 0.5 to 40 nM and with a low detection limit of 0.093 nM (S/N = 3).