Aptamers are single-stranded oligonucleotides with a length of tens of nucleotides and single stranded DNA aptamers have a very highly ordered tertiary structure,which allows them to form stable and specific complex with different targets such as protein,nucleic acid.In this study,we used one of thrombin-binding DNA aptamers to develop a high-sensitive new method to detect proteins based on realtime-PCR and the nature of aptamers and exonuclease I. Three oligonucleotide sequences (probe of thrombin aptamer and two connectors) were synthesized first. When thrombin and its aptamer incubated together, the thrombin bound an aptamer and thereby was protected from degraded by exonuclease I. Subsequently, the protected aptamer probes acted as linkers to join two free connectors, which contained sequences matching the probe. The joined products could reflect the identity and amount of the target protein. Two regression equations of series of ligation product standard and thrombin were built after realtime-PCR and we can quantify the thrombin of samples via them. we found a very high sensitivity in this method and a linear relation between threshold cycles and corresponding logarithms of different standard concentration(y=-2.95x+33.65, R2 =0.990) and between logarithms of ligation product and the corresponding thrombin(y=0.94x-0.29, R2 = 0.998). Furthermore, some optimal reactive conditions including optimal reactive time,of exonuclease 1 and T-4 DNA~ ligase and optimal ratio of aptamer to connector were also defined,respectively.