Here, we report the use of an aptamer-immobilized electrospun polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA) nanofiber as a new aptasensor platform for protein detection. Two thrombin-binding aptamers (TBA29 and TBA15) were used as a model platform to facilitate efficient detection of thrombin in a sandwich manner. Thrombin concentration was measured by fluorescence microscopy and spectroscopy, in which aptamers were labeled with either fluorescein dye or quantum dots. The results indicated that thrombin was captured uniformly on the surface of the nanofiber. Using this sandwich-type biosensor, the minimum detectable concentration of thrombin was 10 pM, with a dynamic range of 0.1-50 nM, when quantum dots were used for labeling. In contrast, the limit of detection was 1 nM, with a dynamic range of 10-200 nM, when using fluorescein dye labeling. This aptamers-on-nanofiber-based biosensor showed 2500-fold higher sensitivity than a 96-microwell plate format, attributed mainly to the large surface area of the nanofibers. In addition, this novel platform also exhibited similar high sensitivity in the detection of exogenously added thrombin in diluted human serum. This aptamers-on-nanofiber system, which is competitive with other sensing platforms and clinically meaningful in terms of its detection limit, is expected to be useful for the detection of various other targets because of its ease of application and manipulation.