In this study, a novel fluorescence resonance energy transfer (FRET) aptasensor for detection of Hg2+ ions in real samples is described. The sensor is based on single-stranded DNA (ssDNA) modified carbon dots (CDs) as energy donor and complementary DNA (cDNA) modified Au nanoparticles (AuNPs) as energy acceptor. The CDs were synthesized from hydrothermal reaction of histidine. The resulting CDs were conjugated with ssDNA through amidation reaction. The AuNPs-cDNA was prepared by self-immobilization of thiolated cDNA on the surface of Au nanoparticles. Specific hybridization between ssDNA and cDNA was achieved by mixing CDs-ssDNA and AuNPs-cDNA. The result of this phenomenon is quenching of the fluorescence associated with the FRET process. The response of the aptasensor is based on selective interaction of Hg2+ ions with thymine (T) groups in CDs-ssDNA, which leads to displacement of AuNPs-cDNA by Hg2+ and an increase in the fluorescence intensity. The aptasensor could be used in the range 1.3 ×10−12 to 2.4 ×10−5 M Hg2+ ion with a detection limits of 7.5 ×10−13 M. It shows high selectivity for Hg2+ with respect to several common metal ions. The aptasensor has been used for the determination of mercury ion concentration in fish samples.