Nanohybrids were formed from 3-mercaptopropionic acid(MPA)-coated Mn-doped ZnS quantum dots(QDs) and methylene blue(MB) via electrostatic interaction, and then used in the detection of trace DNA.The principle of detection is as follows: MB binds with Mn-doped ZnS QDs via electrostatic interaction,and then quenches the room temperature phosphorescence(RTP) of the QDs through photoinduced electron-transfer(PIET). After the addition of DNA, MB binds with DNA through intercalation and electrostatic interaction, and desorbs from the surfaces of Mn-doped ZnS QDs, which recovers the RTP of the QDs. On this basis, a DNA detection method based on the properties of RTP was set up. This method shows a detection range of 0.2–20 mg/L, and a detection limit of 0.113 mg/L. Since this method is based on the RTP of QDs, it is not interfered by the background fluorescence or scattering light in vivo, and thus,avoids complex sample pretreatment. Thus, this method is very feasible for detection of trace DNA in biofluids. Nanohybrids were formed from 3-mercaptopropionic acid (MPA) -coated Mn-doped ZnS quantum dots (QDs) and methylene blue (MB) via electrostatic interaction, and then used in the detection of trace DNA. The principle of detection is as follows: MB binds with electrostatic interaction via Mn-doped ZnS QDs, and then quenched the room temperature phosphorescence (RTP) of the QDs through photoinduced electron-transfer (PIET). After the addition of DNA, MB binds with DNA through intercalation and electrostatic interaction, and desorbs from the surfaces of Mn-doped ZnS QDs, which recovers the RTP of the QDs. On this basis, a DNA detection method based on the properties of RTP was set up. This method shows a detection range of 0.2-20 mg / L, and a detection limit of 0.113 mg / L. Since this method is based on the RTP of QDs, it is not interfered by the background fluorescence or scattering light in vivo, and thus, avoids complex sample pretreatment. Thus, this method is very feasible for detection of trace DNA in biofluids.