The accurate discrimination of microRNAs (miRNAs) with highly similar sequences would greatly facilitate the screening and early diagnosis of diseases. In the present work, a locked nucleic acid (LNA)-modified probe was designed and used for α-hemolysin (α-HL) nanopore to selectively and specifically identify miRNAs. The hybridization of the LNA probe with the target miRNAs generated unique long-lived signals in the nanopore thus facilitated an accurate discrimination of miRNAs with similar sequences, even a single-nucleotide difference. Furthermore, the developed nanopore-based analysis with LNA probe could selectively detect target miRNAs in a natural serum background. This selective and sensitive approach may be highly valuable in the detection of clinically relevant biomarkers in complex samples.