Electrochemical analysis of single molecules is a method with the strong ability of the enhanced efficiency and ultra-sensitivity.Here,we demonstrate that the electrochemical confined space could efficiently convert single molecule characteristics into measurable electrochemical signatures with high temporal resolution.The human telomere repeat sequence T8 was used as a probe to determine the electrochemical confined effect in a nanopore.Our results show that the nanopore with comparable confined space of the telomere repeat sequence exhibits the most distinguishable single-molecule signals which suggest the folded conformation of T8.This method will greatly extend the lifetime of a metastable conformation for a single biomolecule by strong analyte-nanopore interactions,which brings the new insight into the understanding of the biomolecule’s function at single-molecule level. Electrochemical analysis of single molecules is a method with the strong ability of the enhanced efficiency and ultra-sensitivity. Here, we demonstrate that the electrochemical confined space couldfficient convert single molecule characteristics into measurable electrochemical signatures with high temporal resolution. The human telomere repeat sequence T8 was used as a probe to determine the electrochemical confined effect in a nanopore. Our results show that the nanopore with comparable confined space of the telomere repeat sequence exhibits the most distinguishable single-molecule signals which suggest the folded conformation of T8.This method will greatly extend the lifetime of a metastable conformation for a single biomolecule by strong analyte-nanopore interactions, which brings the new insight into the understanding of the biomolecule’s function at single-molecule level.