An aptasensor was designed for sensitive detection of thrombin using in biological fluids by integrating a magnetic aptamer-microbeads. To achieve this goal, the surface of gold plated QCM crystals was coated with L-cysteine and a thrombin binding DNA aptamer was immobilized on the L-cysteine coated QCM crystals surface via glutaraldehyde coupling. The binding interactions of thrombin to QCM crystals were characterized. Magnetic poly(2-hydroxyethyl methacrylate-ethylene glycol dimethacrylate-vinylene carbonate), Mp(HEMA-EGDMA-VC) microbeads were synthesized and thrombin binding aptamer (TBA) was immobilized. The Mp(HEMA-EGDMA-VC)-TBA microbeads were effectively adsorbed thrombin from serum in a relatively short contact time (ca. 5.0 min), and the eluted protein from Mp(HEMA-EGDMA-VC)-TBA was transferred to the QCM aptasensor that showed a specific detection of thrombin from serum. The detection limit of thrombin using aptasensor was 1.00 nmol L-1. The calculation dissociation constant of the aptasensor was 68.5 nmol L-1. The selectivity of the aptasensor system was tested with three different proteins (i.e., elastin, immunoglobulin G (IgG) and human serum albumin (HSA)) and showed high specificity to thrombin. The aptasensor was regenerated by washing with NaOH solution, and repeatedly used until 20 cycles without a change in the performance.