<p>Herein, a sensitive photoelectrochemical (PEC) biosensor was designed for the detection of mercury ion (Hg²⁺) based on deposited gold nanoparticles (DepAu) enhanced titanium dioxide (TiO₂) accompanied by thymine-Hg²⁺-thymine (T-Hg²⁺-T) structure. TiO₂-modified glassy carbon electrode (GCE) exhibited a baseline anodic photocurrent, which was substantially enhanced upon electrochemical deposition of Au NPs. This PEC signal amplification was ascribed to the dual contributions of the improved photon utilization and the facilitated charge transfer. The electrode was then subjected to stepwise modification with T-rich DNA followed by 1-hexanethiol (HT), each incremental layer engendering a concomitant decrement in the photocurrent response. Ultimately, the introduction of target Hg²⁺ enabled specific coordination with the T-rich DNA sequence, facilitating the formation of a stable T-Hg²⁺-T complex architecture on the GCE surface. This coordination event substantially amplified interfacial steric hindrance, thereby impeding electron transfer and effectuating pronounced photocurrent suppression. The quenched PEC response provided a quantitative metric for Hg²⁺ determination. The sensitive and selective detection of Hg²⁺ was achieved in the range of 100 pmol/L to 100 µmol/L, with the detection limit of 33.3 pmol/L. This PEC biosensing platform holds considerable promise for extending to the detection of diverse heavy metal ions and other environmental contaminants by rational design of recognition elements.</p> Graphical abstract <p></p>