<p>Simple, rapid, and sensitive on-site detection methods for Cd²⁺ remain to be developed. Therefore, a label-free fluorescence sensing method based on an aptamer, exonuclease I (Exo I), and SYBR Gold (SG) was established. In the presence of Cd²⁺, the aptamer folds into a stable hairpin structure to protect its 3′ terminus from Exo I cleavage, thereby providing intercalation sites for the fluorescent probe. SG shows significantly stronger fluorescence when bound to intact, base-stacked, hairpin DNA structures than to short digested DNA fragments. After systematic optimization of Exo I concentration, enzymatic hydrolysis time, buffer pH, and SG concentration, a good linear relationship over the Cd²⁺ concentration range of 0–1000 nM with a limit of detection as low as 28.3 nM (3.18 μg/L), and excellent selectivity for Cd²⁺ was established. The method was applied to Cd²⁺-spiked drinking water and rice samples, yielding recoveries of 96.7–106.3% in water and 92.8–103.4% in rice, demonstrating the practicability and reliability of this method.</p>