Heparin(HP) and heparan sulfate(HS) are highly anionic glycosaminoglycans that play essential roles in diverse biological processes through the metal ion-mediated interactions with proteins. However, direct characterization of HS-metal ion interactions at the single-molecule level in solution remains challenging. Nanopore electrochemistry is a label-free and single-molecule technique that enables direct analysis of individual molecular interactions. In this study, a T232K/K238Q Aerolysin nanopore featuring an enhanced electrostatic repelling barrier was utilized to probe the interactions between HS and different metal ions. By systematically varying the electrolyte cations(Na+, K+, and Ca2+), we have found that the metal ions significantly regulate HS translocation behavior by modulating its conformation, charge screening, and HS-nanopore interactions. Notably, in addition to Ca2+, which exhibits strong binding affinity to HS, the monovalent cations Na⁺ and K⁺ with similar physicochemical properties and weaker binding also induce distinct single-molecule signal signatures. Our results demonstrate that the nanopore-based single-molecule analysis holds strong potential to resolve the fine structural features of HS, enabling the characterization of sulfation site distributions, repeat-unit lengths, and related sequence features, and thereby providing a new avenue for high-resolution analysis of complex glycans.