The dual-emission carbon dots (CDs) are used to fabricate an ultra-sensitive and highly selective fluorescence resonance energy transfer (FRET) aptameric-sensor for the quantitation of Hg(II). The designed strategy worked based on hybridization between Trich ssDNA(S1) immobilized on CDs and AuNPs modified with complementary aptamer (AuNPs-S2) and Rhodamine B (RB) as an extra internal reference. Under optimized experimental conditions, the intensity ratio of I580/I668 shows a good linear relationship with the Hg(II) concentration in concentration range of 1.0 × 10−18 to 50.0 × 10−5Mwith detection limit of 5 × 10−19 M. The proposed FRET aptasensor showed high selectivity for Hg(II) determination even in the presence of other metal ions with higher concentration as high as 1000 times. Furthermore, the RB-CDs probe was demonstrated to be efficient for MDA-MB 231 cell imaging. The bifunctional signaling probe exhibits impressive simplicity, convenience, and low detection time and the proposed sensor can be applied in biomedicine study, food safety, and environmental protection.