A high sensitive voltammetric method for rapid determination of thrombin spiked in whole blood by taking advantage of both aptamer-based recognition and the use of a nanoporous membrane has been developed. The nanoporous membrane not only acts as platform for the thrombin recognition but also as ﬁlter of the micrometric components such as white and red blood cells, consequently minimizing matrix effects. The protocol involves a sandwich format in the inner walls (200 nm diameter) of an anodized alumina oxide ﬁlter membrane (AAO). The analytical signal, by DPV oxidation of [Fe(CN)6]4, is based on the blockage in the pores which affects the diffusion of [Fe(CN)6]4 to the screen-printed carbon electrotransducer (SPCEs) modiﬁed with the membrane. By labeling the anti-thrombin IgG with AuNPs followed by silver enhancement a greater passive signal enhancement in comparison to the membrane blockage has been observed. The contribution of both electrostatic/steric effects in this blockage due to the subsequent formation of the aptamer-thrombin complex and the ﬁnal sandwich assay is investigated. The efﬁciency of the system is also monitored by microscopic techniques. The resulted biosensing system allows detecting thrombin spiked in whole blood at very low levels (LOD 1.8 ng mL1) which are within the range of clinical interest for the diagnostic of coagulation abnormalities as well as pulmonary metastasis.