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[Display omitted] • Establishment of a cost-effective nanopore platform for NCL identification at low LOD with trace amount of sample. • Discrimination of single-molecule topologies of DNA/RNA G4 with distinct sequences while closed in sequence length. • Proof of the well-binding properties of NCL with G4 folded from both DNA and RNA. • Recognition of NCL and NCL/G4 complex in human serum. DNA/RNA quadruplexes (G4) are significant secondary structures that exist in human genes, playing essential part in transcription and replication. Nucleolin (NCL) is a ubiquitous protein mainly found in the human nucleus. It is frequently overexpressed in cancer cells and displays a remarkable preference for DNA/RNA G4 binding, playing a crucial role in controlling nucleic acid metabolism and chromatin structure. Additionally, NCL is involved in microRNA (miRNA) biogenesis.This work presents the discrimination of DNA/RNA G4 topologies harboring close sequence length with size-matched nanopore devices. The single-molecule identification of NCL under various parameters was implemented, and a low detection limit of 0.5 nM was achieved, NCL could specifically bind with DNA G4 AS1411 and RNA G4 (rG4) from pre-miRNA 92b and pre-miRNA 149, forming stable complexes that demonstrate distinct nanopore translocation properties. The augmentation of the molar ratio of G4 to NCL induces elevated complex formation. Recognition of both NCL and NCL-G4 complex in human serum is realized. Our work established a precise platform for the differentiation of nucleic acid secondary structure with similar conformation, we also proved the binding behaviors between NCL and DNA/RNA G4, as well as the serum interference test, which is valuable for the investigation of the interactions between proteins and nucleic acids as well as for further understanding the mechanism of the NCL-related diseases processes. [ABSTRACT FROM AUTHOR]