Thiazole Orange (TO)-conjugated Peptide Nucleic Acid (PNA) probes have been reported as a valuable strategy for DNA analysis; however, no investigations targeting RNA molecules and no comparisons between different derivatization approaches have been reported so far. In this work, two TO-conjugated PNAs for genogroup II noroviruses (NoV GII) detection were designed and synthesized. Both the probes target the most conserved stretch of nucleotides identified in the open reading frame 1-2 (ORF1-ORF2) junction region and differ for the dye conjugation strategy: one PNA is end-labelled with the TO molecule tethered by a linker; the other probe bears the TO molecule directly linked to the PNA backbone, replacing a conventional nucleobase. The spectroscopic properties of the two PNA probes were studied and their applicability to NoVs detection, using an isothermal assay, was investigated. Both probes showed good specificity and high fluorescence enhancement upon hybridization, especially targeting RNA molecules. Moreover, the two probes were successfully employed for NoVs detection from stool specimens in an isothermal-based amplification assay targeting RNA 'amplicons'. The probes showed to be specific even in the presence of high concentrations of non-target RNA.