Heavy metal ions, such as thallium(I) and lead(II) are environmental toxicants known to cause a severe threat to human and ecosystem health. This work investigates aptamers and intercalating chromophore-based complexes for the detection of these toxic species. In one method, a selective label-free "turn-on" biosensor was developed using a G-quadruplex-intercalating agent, berberine. Fluorescence, melting temperature (Tm), and circular dichroism analysis confirmed the affinity and selectivity results, illustrating the potential of these aptasensor methods for improving detection limits. These fluorescence assays were found to perform with a detection limit of 3.4 μM for Tl(I) and 0.84 nM for Pb(II). Furthermore, the assays were challenged successfully with Tl(I) and Pb(II) spiked into river water samples. We next developed paper-based fluorescent assays for Tl(I) and Pb(II), where the aptamer/berberine complex was spotted onto the paper test zone. When Tl(I) or Pb(II) ions solutions were spotted onto the top of the test zone and the spot was illuminated with a portable UV light (365 nm), a strong green fluorescence could be easily visualized with the naked eye. The lowest detection limits achieved with these fluorescent paper-based assays for Tl(I) and Pb(II) were 1.1 nM and 1.6 nM, respectively. The two fluorescent approaches presented here have the potential to be the basis of rapid, fast, and cost-efficient screening assays for these toxic species.