Mercury (Hg(2+)) is a highly toxic and widespread environmental pollutant. Herein, a regenerable and highly selective core-shell structured magnetic mesoporous silica nanocomposite with functionalization of thymine (T) and T-rich DNA (denoted as Fe3O4@nSiO2@mSiO2-T-TRDNA nanocomposite) has been developed for simultaneous detection and removal of Hg(2+). In this work, the thymine and T-rich DNA were immobilized onto the interior and exterior surface of outermost mesoporous silica, respectively. The detection mechanism is based on Hg(2+)-mediated hairpin structure formed by T-rich DNA functionalized on the exterior surface of the nanocomposites, where, upon addition of SYBR Green I dye, strong fluorescence is observed. In the absence of Hg(2+), however, addition of the dye results in low fluorescence. The limit of detection for Hg(2+) in a buffer is 2 nM by fluorescence spectroscopy. Simultaneously, the Fe3O4@nSiO2@mSiO2-T-TRDNA nanocomposite features a selective binding with Hg(2+) between two thymines immobilized at the interior surface of the mesopores and exhibits efficient and convenient Hg(2+) removal by a magnet. Kinetic study reveals that the Hg(2+) removal is a rapid process with over 80% of Hg(2+) removed within approximately 1 h. The applicability of the developed nanocomposites is demonstrated to detect and remove Hg(2+) from samples of Xiangjiang river water spiked with Hg(2+). In addition, distinguishing aspects of the Fe3O4@nSiO2@mSiO2-T-TRDNA nanocomposites for Hg(2+) detection and removal also include the regeneration using a simple acid treatment and resistance to nuclease digestion. Similar process can be used to functionalize the Fe3O4@nSiO2@mSiO2 nanocomposites with other nucleic acids and small molecules for environmental and biomedical applications.