Aptamer biosensor for label-free thrombin detection based on a tapered fiber-optic interferometer has been proposed and demonstrated. The biosensor probe can be easily fabricated by tapering a commercial double-cladding fiber down to 5 μm in diameter and it provides a high refractive index sensitivity of 1660 nm/RIU. The changes are determined by tracing the wavelength shift of the dip in the transmission spectrum with variable surrounding refractive index. With the help of the surface functionalization of thrombin-binding aptamers, the proposed fiber-optic biosensor proves its capability in label-free detection of thrombin with a lowest detectable concentration of 0.1 μm, together with high specificity in various concentrations of pure and mixed thrombin solutions.