A highly sensitive and selective photoelectrochemical (PEC) biosensor for Hg(2+) detection was developed on the basis of the synergistic effect of exciton energy transfer (EET) between CdS quantum dots (QDs) and Au nanoparticles (NPs) coupled with sensitization of rhodamine 123 (Rh123) for signal amplification. First, the TiO2/CdS hybrid structure obtained by depositing CdS QDs on TiO2 film was employed as a matrix for immobilizing probe DNA (pDNA). Next, Rh123 was introduced into the pDNA terminal, and then Au NP labeled target DNA (Au-tDNA) was hybridized with pDNA to form a rod-like double helix structure. The detection of Hg(2+) was based on a conformational change of the pDNA after incubating with Hg(2+). In the absence of Hg(2+), Rh123 was located away from the electrode surface due to the DNA hybridization, leading to inhibition of the sensitization effect, and meanwhile, the occurrence of EET between CdS QDs and Au NPs resulted in a photocurrent decrease. However, after incubating with Hg(2+), the rod-like double helix was disrupted, and the energy transfer was broken. In this case, the photocurrent recovered, and meanwhile, the folded pDNA made the labeled Rh123 move closer to the electrode surface, leading to the formation of the sensitization structure, which evidently increased the photocurrent intensity. The sensitivity of the biosensor for Hg(2+) detection was greatly enhanced for the dual signal amplification strategy. The linear range was 10 fM to 200 nM, with a detection limit of 3.3 fM. This biosensor provides a promising new platform for detecting various heavy metal ions at ultralow levels.