The precise control of the conformations of biomolecules adsorbed on a surface at the single-molecule level is significant. However, it remains a huge challenge because of the complex structure and conformation diversity of biomolecules. Herein, a "nanopore-confined recognition" strategy is proposed to manipulate the adsorption of individual valinomycin molecules at room temperature through precise design of functionalized conjugated macrocycle (CPN8) supramolecular nanopores with complementary architectures and binding sites. We revealed that CPN8 prefers to selectively recognizing valinomycin with complementary architecture because of the strong synergistic interactions between the isopropyl groups of valinomycin and the amino groups of CPN8, with valinomycinhighly oriented pyrolytic graphite (HOPG) interactions. Our perspectives at the single-molecule level will provide valuable insights to improve the design of supramolecular nanopores for conformation-selective recognition of non-conjugated molecules.