The need for rapid and sensitive diagnostic tools is emphasized by the significant impact of infectious diseases on global health. This study presents a cell-free biosensor utilizing toehold switch technology, combined with nucleic acid sequence-based amplification (NASBA), for high specificity and sensitivity in Zika virus detection. The toehold switch, a denovo-designed regulator of gene expression, forms the crux of our detection system, offering a versatile and programmable approach to nucleic acid-based diagnostics. The cell-free system based on