With 3-mercaptopropionic acid as the stabilizer, Mn-doped ZnS quantum dots (QDs) are synthesized via the water phase method. These QDs emit strong phosphorescence at room temperature. These QDs aggregate with cetyl trimethylammonium bromide (CTAB) as a cation surfactant through electrostatic interaction to form Mn-doped ZnS QDs/CTAB nanohybrids, thereby largely enhancing the room-temperature phosphorescence (RTP) of QDs. The newly-added mitoxantrone (MXT) binds with CTAB through hydrophobic interaction and structure function to form more stable mixture, leading to separation of CTAB from the QD surfaces and reduction of the QD RTP intensity. The results indicate the nanohybrids can significantly improve the QD-based MXT detection ability. Thereby, a high-performance and high-sensitivity RTP sensor for MXT detection is established. Under the optimal conditions, this sensor has a MXT detection limit of 0.23 nmol/L and linear range from 0 to 200 nmol/L (correlation coefficient R=0.99). The results with real urine and serum samples are with recovery rate of 98.6%～102.5%. The phosphorescence detection method is simple, rapid, sensitive and selective, and can be used well for analysis and detection of MXT in body fluid.