We have isolated two DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF165). To select aptamers that have high specificity for VEGF165, we used another protein as a competitor in the SELEX process. In addition, we evaluated the screened library with aptamer blotting. Isolated aptamers bound to VEGF165 with Kd in a few hundred-nanomolar ranges, and did not bind to a competitor. This selection method allowed us to select the specific aptamers against a target protein efficiently. Furthermore, isolated aptamers did not bind to VEGF121. These specific DNA aptamers against VEGF165 would be a useful sensing element for cancer diagnosis.