A new solid-state, Al2O3 nanopore sensor with enhanced surface properties for the real-time detection and analysis of individual DNA molecules is reported. Nanopore formation using electron-beam-based decomposition transforms the local nanostructure and morphology of the pore from an amorphous, stoichiometric structure (O to Al ratio of 1.5) to a heterophase crystalline network, deficient in O (O to Al ratio of ≈0.6). Direct metallization of the pore region is observed during irradiation, thereby permitting the potential fabrication of nanoscale metallic contacts in the pore region with application to nanopore-based DNA sequencing. Dose-dependent phase transformations to purely γ and/or α-phase nanocrystallites are also observed during pore formation, allowing for surface-charge engineering at the nanopore/fluid interface. DNA transport studies reveal an order-of-magnitude reduction in translocation velocities relative to alternate solid-state architectures, accredited to high surface-charge density and the nucleation of charged nanocrystalline domains. The unique surface properties of Al2O3 nanopore sensors make them ideal for the detection and analysis of single-stranded DNA, double-stranded DNA, RNA secondary structures, and small proteins. These nanoscale sensors may also serve as useful tools in studying the mechanisms driving biological processes including DNA–protein interactions and enzyme activity at the single-molecule level.