Nonprotein coding RNA (npcRNA) is a transcribed gene sequence that is not able to translate into protein, yet it executes a specific function in modulation and regulation mechanisms. As npcRNA is highly resistant to the mutation, the Sau-02 npcRNA gene and its probe oligonucleotide, which are specifically present in Staphylococcus aureus and in methicillin-resistant S. aureus only, used to develop a highly specific and sensitive colorimetric assay on unmodified gold nanoparticles (AuNPs). Hybridization between the npcRNA Sau-02 gene sequences was detected through noncrosslinking AuNP aggregation in salt solution in the presence of probe-target gene sequences. AuNPs of 10 and 15 nm in sizes with monovalent ion salt (NaCl) solution were optimized as the ideal tool for investigating the stability of AuNPs upon the addition of gene sequences. The state dispersed and aggregated forms of 10 nm AuNPs with the presented colorimetric assay were justified through field emission scanning electron microscopy and atomic force microscopy. The particle distribution of two different AuNP states was evaluated through particle distribution analysis. The lowest detection amount of S. aureus npcRNA from the colorimetric assay performed was 6 pg/µL, as the color of AuNPs turned blue with the presence of probe oligonucleotides and target gene sequences.