This work describes a new nanosensor for the simple, rapid, portable, colorimetric analysis of mercury(II) (Hg²⁺) ions by combining the sensitive Tyndall effect (TE) of colloidal Au nanoparticles (AuNPs) with specific thymine–Hg²⁺–thymine (T–Hg²⁺–T) coordination chemistry for the first time. For the TE-inspired assay (TEA), in the presence of Hg²⁺ in a sample, the analyte can selectively mediate the hybridization of three types of flexible single-stranded DNAs (ssDNAs) to form stable rigid double-stranded DNAs (dsDNAs) via the T–Hg²⁺–T ligand interaction. Subsequent self-assembly of the dsDNAs with terminal thiol groups on the AuNPs' surfaces led to their “double” aggregation in addition to the lack of sufficient ssDNAs as the stabilizing molecules in a high-salt solution, resulting in a remarkably enhanced TE signal that positively relied on the Hg²⁺ level. The results demonstrated that such a TEA method enabled rapid naked-eye qualitative analysis of 625 nM Hg²⁺ within 10 min with an inexpensive laser pointer pen as an inexpensive handheld light source to generate the TE response. Making use of a smartphone for portable TE readout could further quantitatively detect the Hg²⁺ ions in a linear concentration range from 156 to 2500 nM with a limit of detection as low as 25 nM. Moreover, the developed equipment-free nanosensor was also used to analyze the Hg²⁺ ions in real samples including tap water, drinking water, and pond water, the obtained recoveries were within the range of 93.68 to 108.71%. To the best of our knowledge, this is the first report of using the AuNPs and functional nucleic acids to design a TE-based biosensor for the analysis of highly toxic heavy metal ions.