A ratiometric fluorescent sensor for mercury ions (Hg2+) has been constructed via covalent functionalization of silicon nanodot (SiND) with Hg2+-specific 6-carboxy-X-rhodamine (Rox)-tagged DNA. For the Rox-DNA functionalized SiND, the red fluorescence of Rox can be quenched by the blue-emitting SiND in the presence of Hg2+ due to structural change in DNA, which serves as the response signal. Meawhile, the fluorescence of SiND is insensitive to Hg2+ and acts as the reference signal. The wavelength difference in the optimal emission peak is as large as 190 nm between SiND (422 nm) and Rox (612 nm), which can efficaciously exclude the interference of the two emission peaks, and facilitates dual-color visualization of Hg2+ ions. The biofunctionalization of SiND improves the acid-base stability of SiND significantly, which is favorable for its application in the intracellular environment. Accordingly, a sensitive, simple, precise and rapid method for tracing Hg2+ was proposed. The limit of detection and precision of this method for Hg2+ was 9.2 nM and 8.8% (50 nM, n = 7), respectively. The increase of Hg2+ concentration in the range of 10-1500 nM was in accordance with linearly increase of the I422/ I612 ratio. As for practical application, the recoveries in spiked human urine and serum samples were in the range of 81-107%. Moreover, this fluorescent nanosensor was utilized to the ratiometric detection of Hg2+ in HeLa cells.