The hybridization of single-stranded oligonucleotide-derivatized gold nanoparticles (Au nanoprobes) with double stranded complementary DNA was directly observed by atomic force microscopy (AFM). This specific interaction is the basis for an Au nanoprobe-based homogeneous assay for specific DNA sequence detection, based on salt-induced particle aggregation that is prevented when a complementary target is present. For long DNA targets (linearized plasmid DNA) complicated hybridized target DNA-Au-nanoprobes structures were formed, that were interpreted as the basis for stability of the Au nanoprobes against salt-induced aggregation. For shorter DNA targets (PCR amplified fragments) hybridization with the Au nanoprobes occurred, in the majority of cases, in the expected location of the DNA target fragment containing the specific sequence. The formation of the observed DNA hybridized structures provides evidence at the molecular level for specific hybridization to the target sequence as the method of binding of the Au nanoprobes.