Using the bi-cell surface plasmon resonance instrument, we were able to detect thrombin in the concentration range of 500 femtomoles to 2 picomoles with thrombin aptamer as the capture probe. The lowest limit of detection was 250 femtomoles. The affinity of the aptamer for prothrombin was also investigated, which was detected at 2 picomoles. Using serum samples, 1 picomole of thrombin was detected. We proved that an aptamer could be used as a capture ligand in bi-cell SPR instrument. This model could be used in a clinical setting for the real time detection of diagnostically relevant biomolecules.