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Enteroviruses are ubiquitous mammalian pathogens that can produce mild to life-threatening disease. We developed a multimodal, rapid, accurate and economical point-of-care biosensor that can detect nucleic acid sequences conserved amongst 96% of all known enteroviruses. The biosensor harnesses the physicochemical properties of gold nanoparticles and oligonucleotides to provide colourimetric, spectroscopic and lateral flow-based identification of an exclusive enteroviral nucleic acid sequence (23 bases), which was identified through in silico screening. Oligonucleotides were designed to demonstrate specific complementarity towards the target enteroviral nucleic acid to produce aggregated gold–oligonucleotide nanoconstructs. The conserved target enteroviral nucleic acid sequence (≥1 × 10−7 M, ≥1.4 × 10−14 g/mL) initiates gold–oligonucleotide nanoconstruct disaggregation and a signal transduction mechanism, producing a colourimetric and spectroscopic blueshift (544 nm (purple) > 524 nm (red)). Furthermore, lateral-flow assays that utilise gold–oligonucleotide nanoconstructs were unaffected by contaminating human genomic DNA, demonstrated rapid detection of conserved target enteroviral nucleic acid sequence (