Abstract: Accurate quantification of various mercury (Hg) species dynamics in groundwater is critical for understanding Hg mobilization, fate, and consequent impacts on water ecological security. This foundational work, however, faces challenges due to the lack of highly sensitive, reliable, and field-deployable detection technologies that can determine and monitor ultra-trace Hg(II) in groundwater. Here, this research presents and assesses two types of biosensing methods for dissolved Hg(II) based on a deoxyribonucleic acid (DNA) sensing material: the DNA-functionalized hydrogel for direct Hg(II) detection in groundwater and the DNA-DGT sensor for simultaneous sampling and detection with the diffusive gradients in thin films (DGT) technique. Applying tests to hydrogeochemically diverse groundwaters from the Grand River Watershed, Canada, the results indicate that the DNA-functionalized hydrogel is able to quickly detect dissolved Hg(II) but inapplicable to low Hg(II) concentrations (<1.60 μg/L), whereas the DNA-DGT sensor can capture variably ultra-trace Hg(II) species depending on the deployment time. Quantification of Hg(II) species in groundwater via joint DNA-DGT sensing and hydrogeochemical calculation indicates that temperature, pH, Cl⁻, and dissolved organic matter significantly affected partitioning of trace Hg(II) between various mobile species, diffusion efficiency, and thus its mobility. Combined with hydrogeochemical modeling, the DNA-DGT measurements reveal that mobilization and transformation of Hg(II) are linked to redox cycling of sulfur in groundwater. This study therefore highlights that monitoring of low-level Hg(II) with ultra-sensitive, field-deployable biosensing methods is of significance to understanding mobility and fate of Hg in groundwater and its threat to safe drinking water supply.