A fluorescent probe was developed for ratiometric detection of thallium ions in mineral water samples by modifying a G-rich aptamer (PS2.M − 7) with a fluorescence donor (Cyanine-3, Cy3) and a quencher (Cyanine-5, Cy5). The probe had a random coil structure that changed into a G-quadruplex structure upon binding with Tl+. This change in structure decreased the distance between the donor and acceptor moieties, which resulted in fluorescence resonance energy transfer between Cy3 and Cy5. Under optimized conditions, the limit of detection and linear concentration range for Tl+ were 30.1 μM (3σ) and 10 μM–10 mM (R2 = 0.9981), respectively. This simple and cost-effective fluorescence sensor provided satisfactory results for detection of thallium ions in spiked mineral water samples.