Among the various types of post-translational modifications (PTMs), methylation is the simple functionalized one that regulates the functions of proteins and affects interactions of protein-protein and protein-DNA/RNA, which will further influence diverse cellular processes. The methylation modification has only a slight effect on the size and hydrophobicity of proteins or peptides, and it cannot change their net charges at all, so the methods for recognizing methylated protein are still limited. Here, we designed a recognition receptor consisting of a α-hemolysin (α-HL) nanopore and polyamine decorated γ-cyclodextrin (am8γ-CD) to differentiate the methylation of peptide derived from a heterogeneous nuclear ribonucleoprotein at the single molecule level. The results indicate that the modification of a methyl group enhances the interaction between the peptide and the recognition receptor. The results of molecular simulations were consistent with the experiments; the methylated peptide interacts with the receptor strongly due to the more formation of hydrogen bonds. This proposed strategy also can be used to detect PTM in real biological samples and possesses the advantages of low-cost and high sensitivity and is label-free. Furthermore, the success in the construction of this recognition receptor will greatly facilitate the investigation of pathogenesis related to methylated arginine.