A novel fluorescence aptasensor based on DNA charge transport for sensitive protein detection has been developed. A 15nt DNA aptamer against thrombin was used as a model system. The aptamer was integrated into a double strand DNA (dsDNA) that was labeled with a hole injector, naphthalimide (NI), and a fluorophore, Alexa532, at its two ends. After irradiation by UV light, the fluorescence of Alexa532 was bleached due to the oxidization of Alexa532 by the positive charge transported from naphthalimide through the dsDNA. In the presence of thrombin, the binding of thrombin to the aptamer resulted in the unwinding of the dsDNA into ssDNA, which led to the blocking of charge transfer and the strong fluorescence emission of Alexa532. By monitoring the fluorescence signal change, we were able to detect thrombin in homogeneous solutions with high selectivity and high sensitivity down to 1.2 pM. Moreover, as DNA charge transfer is resistant to interferences from biological contexts, the aptasensor can be used directly in undiluted serum with similar sensitivity as that in buffer. This new sensing strategy is expected to promote the exploitation of aptamer-based biosensors for protein assays in complex biological matrixes.