In this study, an ultra-sensitive and selective Thrombin biosensor with aptamer-recognition surface is introduced based on carbon nanocomposite. To prepare the this biosensor, screen-printed carbon electrodes (SPCE) were modified with a nanocomposite made from fullerene (C60), multi-walled carbon nanotubes (MWCNTs), polyethylenimine (PEI) and polymer quantum dots (PQdot). The unique characteristics of each component of the C60/MWCNTs-PEI/PQdot nanocomposite allow for synergy between nanoparticles while polymer quantum dots resulted in characteristics such as high stability, high surface to volume ratio, high electrical conductivity, high biocompatibility, and high mechanical and chemical stability. The large number of amine groups in C60/MWCNTs-PEI/PQdot nanocomposite created more sites for better covalent immobilization of amino-linked aptamer (APT) which improved the sensitivity and stability of the aptasensor. Differential Pulse Voltammetry (DPV) method with probe solution was used as the measurment method. Binding of thrombin protein to aptamers immobilized on the transducer resulted in reduced electron transfer at the electrode/electrolyte interface which reduces the peak current (IP) in DPV. The calibration curve was drawn using the changes in the peak current (ΔIP),. The proposed aptasensor has a very low detection limit of 6 fmol L−1, and a large linear range of 50 fmol L−1 to 20 nmol L−1. Furthermore, the proposed C60/MWCNTs-PEI/PQdot/APT aptasensor has good reproducibility, great selectivity, low response time and a good stability during its storage. Finally, the application of the proposed aptasensor for measuring thrombin on human blood serum samples was investigated. This aptasensor can be useful in bioengineering and biomedicine applications as well as for clinical studies.