Biological nanopores provide a unique single-molecule sensing platform to detect target molecules based on their specific electrical signatures. The γ-hemolysin (γ-HL) protein produced by Staphylococcus aureus is able to assemble into an octamer nanopore with a ∼2.3 nm diameter β-barrel. Herein, we demonstrate the first application of γ-HL nanopore for DNA structural analysis. To optimize conditions for ion-channel recording, the properties of the γ-HL pore (e.g., conductance, voltage-dependent gating, and ion-selectivity) were characterized at different pH, temperature, and electrolyte concentrations. The optimal condition for DNA analysis using γ-HL corresponds to 3 M KCl, pH 5, and T = 20 °C. The γ-HL protein nanopore is able to translocate dsDNA at about ∼20 bp/ms, and the unique current-signature of captured dsDNA can directly distinguish guanine-to-inosine substitutions at the single-molecule level with ∼99% accuracy. The slow dsDNA threading and translocation processes indicate this wild-type γ-HL channel has potential to detect other base modifications in dsDNA.