In this paper, a nucleic acid dye-enhanced electrochemical biosensor based on a screen-printed carbon electrode (SPCE) modified with Au nanoparticles (AuNPs) was designed for the detection of Hg²⁺ in water. AuNPs were modified on the surface of the disposable SPCE through the electrodeposition of HAuCl₄. Subsequently, thiolated DNA probes were immobilized on the AuNP-modified electrode surface by Au–S reaction. After Hg²⁺ was bound with a DNA probe by thymine (T)–Hg²⁺–thymine (T) mismatch, the DNA probe was folded into a hairpin structure where positively charged GelRed molecules were embedded into the double-stranded part of the hairpin. Thus, the current of [Fe(CN)₆]^3−/4− increased significantly on account of the decreased electrostatic repulsion at the electrode surface. Under the optimized experimental conditions, the peak current of [Fe(CN)₆]^3−/4− exhibited a good linear relationship with lgC_Hg²⁺ in the concentration of Hg²⁺ linear range of 0.1 nM to 500 nM, and the limit of detection (S/N = 3) was calculated as 0.04 nM. The electrochemical sensor also exhibited excellent selectivity for Hg²⁺ in the presence of nine interfering ions, including Na⁺, Fe³⁺, Ni²⁺, Mg²⁺, Co²⁺, Pb²⁺, K⁺, Al³⁺ and Cu²⁺. Meanwhile, the developed electrochemical sensor was tested in the analysis of Hg²⁺ in tap water and river water samples, and the recoveries ranged from 81.0 to 114%. Therefore, this nucleic acid dye-enhanced electrochemical biosensor provided the advantages of simplicity, sensitivity, and specificity and is expected to be an alternative for Hg²⁺ detection in actual environmental samples.