The direction synthesis of biofunctional nanomaterials with DNA as the template is of high application value. By using phosphorothioate-thymine single-stranded DNA (PS-T-ssDNA) as the template and through synthetic conditions optimization, novel low-toxicity and environment-friendly ssDNA-functionalized room-temperature phosphorescent quantum dots (PS-T-ssDNA RTP QDs) were prepared at low temperature (37 °C). Then, the quantitative RTP-based mercury(II) (Hg2+) detection was achieved by utilizing the specific identifying ability of T-base-pair Hg2+ (T-Hg2+-T) and its photoinduced electron transfer. This RTP sensor in Hg2+ detection had a linear range of 0.02 to 0.8 μM and a detection limit of 4.8 nM. The dependence on RTP of QDs effectively avoids interference from background fluorescence and scattering light in the environment or biological samples. This sensor also possessed an RTP stability and a long service life and did not require sample pretreatment. Thus this sensor is suitable for environmental and quantitative Hg2+ detection in biological samples.