Label-free immunoassay systems have the advantages of procedural simplicity and a low construction cost of surfaces for immunosensing. When label-free immunoassay systems are considered, the nonspecific adsorption of unwanted materials should be eliminated unless it aids in the detection of error. PEG is well-known as a blocking agent for the prevention of the adsorption of nonspecific binding materials when coimmobilized with ligands for targets such as antibodies and oligonucleotides. The construction strategy for PEG/ligand coimmobilized surfaces is an important point in the preparation of a high-performance assays because the physiological condition of the ligand depends strongly on its interaction with the PEG chain. In this report, we investigate the interaction between thrombin and a thrombin binding aptamer (TBA) on a PEG/TBA coimmobilized surface by using a shear horizontal surface acoustic wave (SAW) sensor. The thrombin TBA binding property shows remarkable differences with changes in the PEG density and the distance from the gold surface to the aptamer.