The conical glass nanopore was modified through layer-by-layer electrostatic deposition of a monolayer of glucose oxidase, and then an ultrathin gold film was formed in situ through enzyme-catalyzed reactions. The morphology and components of single glass nanopore before and after ultrathin Au deposition were characterized by transmission electron microscopy (TEM) and energy-dispersive X-ray (EDX) analysis, respectively. In particular, the quenching of the quantum dots fluorescence in the nanopore tip zone further illustrated that the gold nanofilm was successfully deposited on the inner wall of the single glass nanopore. The Au thin films make the glass nanopores more biologically friendly and allow the nanopores facile functionalization of the surface through the Au-S bonds. For instance, the ionic current rectification (ICR) properties of the gold-decorated glass nanopores could be switched readily at different pHs by introducing different thiol molecules.