[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] The nanopore sensor can detect cancer-derived nucleic acid biomarkers such as microRNAs (miRNAs), providing a noninvasive tool potentially useful in medical diagnostics. However, the nanopore-based detection of these biomarkers remains confounded by the presence of numerous other nucleic acid species found in biofluid extracts. Their nonspecific interactions with the nanopore inevitably contaminate the target signals, reducing the detection accuracy. Here we report a novel method that utilizes a polycationic peptide-PNA probe as the carrier for selective nucleic acid detection in the nucleic acids mixture. The cationic probe hybridized with DNA or RNA forms a dipole complex, which can be captured by the pore using a voltage polarity that is opposite the polarity used to capture negatively charged nucleic acids. As a result, non-target species are driven away from the pore opening, and the target sequences can be detected accurately without interference. In addition, we demonstrate that the PNA probe enables to accurately discriminate single-nucleotide difference. Moreover, molecule dynamic simulation is applied to expose the mechanism. Combined with experimental and calculating data, we construct a model to demonstrate that it is universal for all kinds of nucleic acid targets. In sum, this highly sensitive and selective nano-dielectrophoresis approach can be applied to the detection of clinically relevant nucleic acid fragments in complex samples and fulfills the diagnostic of diseases in early stage.