Solid-state nanopores have been employed as useful tools for single molecule analysis due to their advantages of easy fabrication and controllable diameter, but selectivity is always a big concern for complicated samples. In this work, functionalized magnetic core–shell Fe3O4–Au nanoparticles, which acted as a molecular carrier, were introduced into nanopore electrochemical system for microRNA sensing in complicated samples with high sensitivity, selectivity and signal-to-noise ratio (SNR). This strategy is based on the specific affinity between neutral peptide nucleic acids (PNA)-modified Fe3O4–Au nanoparticles and negative miRNA, and the formation of negative Fe3O4–Au–PNA–miRNA complex, which can pass through the nanopore by application of a positive potential and eliminate neutral Fe3O4–Au–PNA complex. To detect miRNA in complicated samples, a magnet has been used to separate Fe3O4–Au–PNA–miRNA complex with good selectivity. We think this is a facile and effective method for the detection of different targets at single molecular level, including nucleic acids, proteins, and other small molecules, which will open up a new approach in the nanopore sensing field.