A novel electrogenerated chemiluminescence (ECL) assay for sensitive determination of thrombin is designed employing CdSe/ZnS quantum dots served as an ECL label. This ECL sensor is fabricated on graphene modified glassy carbon electrode which is then covered with a low surface coverage of gold nanoparticles (AuNPs). An aptamer is used to selectively recognize the target. The thiol-terminated aptamer is first immobilized on AuNPs/graphene modified electrode, and then thrombin is imported to form the aptamer-thrombin complexes. After blocking the nonspecifically bound oligonucleotides with MCH solution, another CdSe/ZnS quantum dots modified aptamer is hybridized with the free thiol-terminated aptamer to form a DNA complexe. A decreased ECL signal is observed upon recognition of the target thrombin. The integrated ECL intensity versus the concentration of thrombin is linear in the range from 0.01 to 50 nM. The detection limit is 10 fM. The present aptasensor also exhibits excellent selectivity, stability and reusability. This sensing system can provide a promising label-free model for aptamer-based compounds sensitive detection.