Though rapid tests were developed for mass screening of prion diseases in the last century, bovine spongiform encephalopathy (BSE) was still epidemic in some European countries. The main reason is that the sensitivity of such tests is insufficient for detecting animals that are incubating with prion diseases at the presymptomatic stage. Driven by this, in this contribution, we developed a novel sensitive label-free method taking advantage of DNA aptamer for prion proteins (PrP) detection through the formation of T-Hg2+-T configuration. In the presence of Hg2+ ions, double-strand structures formed due to the strong binding affinity of Hg2+ ions to the T bases of DNA aptamer, which dramatically enhanced the fluorescence of Syber Green I, a double-strand indicator. With the addition of prion protein, however, the specific interaction between prion protein and its aptamer forced the destruction of the double-strand structures, and thus the fluorescence of Syber Green I decreased. It was found that there is a linear relationship between the decreased fluorescence intensities and prion protein concentration ranging from 13.0 to 156.0 nmol/L. Compared with other methods, the method presented here holds the advantages of being label-free, rapid, highly sensitive, and selective, which shows great promise for clinical application.