Recently, aptamers have been considered as a great alternative to antibodies to develop biosensors because aptamers can strongly bind a specific target molecule. And the aptamer shows the possibility for the commodity industry in developing biosensor because of artificial receptors. Using these aptamers, therefore, many aptamer based electrochemical biosensors have been studied by applying new materials and surface treatment to substrates. However, comparing degree of the biosensor sensitivity in the same aptamers according to the each condition of electrode substrates was still not reported. In this study, we have studied on the sensitivities by changing the experimental conditions for area, material and surface treatment of electrode substrates to develop aptamer based biosensors using thrombin DNA aptamers. The results indicate that the larger exposed area of electrodes is more efficient than the smaller. Moreover, the sensitivity in the screen-printed carbon electrode and GNP/swCNT/mwCNT treated carbon electrode showed the remarkable efficiency than in gold or platinum electrodes. We believe this study can be employed as reference data for developing the commercial aptamer based biosensor.