A signal-amplified lateral flow test strip (SA-LFTS) for the detection of Cu2+ in aqueous solution was constructed based on Cu+-catalyzed click chemistry and hybridization of single-stranded DNA (ssDNA). Alkyne and azide modified ssDNA acted as specific elements for Cu2+ recognition, and a chemical ligation product formed through Cu+-catalyzed alkyne-azide cycloaddition. Hybridization of ssDNA-labeled gold nanoparticles resulted in high sensitivity, and the output signal could be observed directly by the naked eye. Using the developed SA-LFTS under optimal conditions, Cu2+ could be detected rapidly with limit of detections of 5 nM and 4.2 nM by visual observation and quantitative analysis, respectively. The sensitivity (i.e. the visual limit of detection) of the SA-LFTS was 80-times higher than that of traditional LFTS. The SA-LFTS was applied to the determination of Cu2+ in municipal water and river water samples with the results showing good recovery and accuracy. The developed test strip is promising for point-of-care applications and detection of Cu2+ in the field.