A multi-technique approach to prove the preparation of poly(3,4-ethylenedioxythiophene/cucurbit[7]uril) pseudorotaxanes (PEDOT∙CB7-PPs) is reported. Molecular docking simulation and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) validate the complexation ability of the CB7 molecule towards 3,4-ethylenedioxythiophene (EDOT), which leads to the EDOT∙CB7 inclusion complex. Oxidative polymerization of EDOT∙CB7 enabled the synthesis of PEDOT∙CB7-PPs. The water-soluble part of PEDOT∙CB7-PPs was selected, freeze-dried, and chemically characterized. Furthermore, dynamic light scattering (DLS) has been used to study the particle size and z-potential (ZP-ζ) of PEDOT∙CB7-PPs. The ZP-ζ value (35 mV) evidenced that the PEDOT∙CB7-PPs formed stable water dispersion. By combining the emerging nanopore resistive pulse sensing technique (Np-RPS) and computational modeling, we identified strong interactions of PEDOT∙CB7-PPs with the aerolysin (Ael) nanopore. PEDOT∙CB7-PPs behave as positive charged species, and thus trans negative bias promotes its interactions with the Ael nanopore. The computational modeling results are fully consistent with the Np-RPS detection, which also reveals strong interactions between PEDOT∙CB7-PPs and the Ael nanopore. With this study, we hope to provide new insights and a better understanding of the interactions between supramolecular complexes based on CB7 and biological entities, which is instrumental for future applications in the field of nanobiotechnology.