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Accurate signal quantification is a critical parameter for precise pathogen diagnosis. Furthermore, the development of novel diagnostic methods amenable for portable or scalable purposes may facilitate the real-time surveillance of emerging pathogenic threats. Here, we extrapolated the use of a FokI-driven signal amplification approach, powered by the nickase activity of the FokI restriction endonuclease, to enhance the signal detection of multiple respiratory viral pathogens relevant to human health. This approach utilises a set of dumbbell-like fluorescent oligonucleotides against three families of human single-stranded RNA respiratory viruses (Coronaviridae, Paramyxoviridae, and Pneumoviridae), including human betacoronaviruses (OC43/HKU1), human betacoronavirus SARS-CoV-2, human parainfluenza viruses (HPIV1/HPIV3/HPIV2/HPIV4), and human metapneumovirus (HMPV)/respiratory syncytial virus …