This study presents an ultra-sensitive aptasensor for mercury (Hg2+) ion detection. The Langmuir-Blodgett (LB) technique was utilized for large-scale monolayer deposition of gold nanoplates (GNPlts). Deposition parameters, including barrier speed (5 mm/min), substrate lifting speed (1 mm/min), and surface pressure (70 mN/m), were optimized to ensure uniform film formation. Deposited gold nanoplates were functionalized with a thymine-rich primary aptamer (p-Ap) to form the GNPlts@p-Ap complex, followed by hybridization with a complementary secondary aptamer conjugated to thionine-coated gold nanoparticles (s-Ap-GNP@Th). When Hg2+ was added to the final probe (GNPlts@ds-Ap-GNP@Th), the s-Ap-GNP@Th was released due to the formation of T-Hg2+-T complexes. The concentration of Hg2+ ions was monitored via multi-modal detection using fluorescence spectroscopy, UV–Vis absorption, and smartphone-based colorimetric analysis. The aptasensor exhibited excellent sensitivity, selectivity, speed, and cost-effectiveness. The achieved limit of detection (LOD) for Hg2+ was 5 × 10−7 ppb, with a linear range of 1 × 10−6 to 10 ppb (R2 > 0.98). The unique GNPlts enhanced substrate adhesion, ensuring sensor stability.
