In this work, the investigation of thrombin concentration using microresonator functionalized with thiol-modified thrombin-binding aptamer (TBA) is reported. Resonator is a ball shaped structure on a tip of an optical fiber. Thrombin is a serine protease that plays an important role during blood coagulation; therefore, it is substantial to detect its levels in blood of the patient. The structure has been manufactured using standard single-mode fibers on CO2 laser splicing system. Further, the resonator of diameter 518 μm has been calibrated with sucrose for refractive index changes with optical backscatter reflectometer. Changes in sensitivity and reflectivity in wavelength shift and amplitude fluctuation were measured. This was followed by coating the resonator with gold and functionalizing surface with TBA. Following aptamer immobilization, the performance of 518 μm resonator was examined in different concentrations of thrombin protein from 4.01 nM to 66.84 nM. We report here a sample presenting a sensitivity of (122.65 nm/RIU, RIU = refractive index units), which allows thrombin detection with an average wavelength shift of 1.034 nm.