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In this work, a simple room-temperature phosphorescence (RTP) method was first proposed to detect adriamycin, by using a functional material of Mn doped ZnS quantum dots (Mn-ZnS QDs) composited with poly(diallyldimethylammonium chloride) (PDDA). After PDDA was associated to the Mn-ZnS QDs, the RTP intensity was significantly enhanced. As a result, Mn-ZnS QDs/PDDA nanoassemblies greatly improve the adriamycin detection ability of QDs and provide an important method for developing more effective and sensitive adriamycin detection sensor. The method based upon RTP has a linear range from 0.5 to 64.0 μM with a detection limit (3σ/s) for adriamycin of 0.45 μM. The developed method was further successfully applied to detect adriamycin in human serum samples (diluted 50 times) with satisfactory results, and the recovery ranged from 98.3 to 101.3.