Graphical abstract Highlights • An ultrasensitive phosphorescence turn-on sensing system was fabricated to selectively detect lysozyme in humoral samples.. • This is first attempt to detect lysozyme combining aptamer and room temperature phosphorescence technique. • In this sensing platform, Mn-doped ZnS quantum dots was used as the phosphorescence reporter and lysozyme-aptamer as the recognition unit. • The tedious deoxidization and organization media protection for the phosphorescence measurement is avoided. Abstract An ultrasensitive phosphorescence sensing system was fabricated to selectively detect lysozyme in humoral samples engaging Mn-doped ZnS quantum dots as the room temperature phosphorescence reporter and lysozyme-aptamer as the recognition unit. The phosphorescence emission of Mn-doped ZnS quantum dots was quenched by lysozyme-aptamer on account of the supramolecular interaction between them. When lysozyme was introduced into the system, it was preferred to form lysozyme-aptamer/lysozyme complex and the lysozyme-aptamer was released from the surface of the quantum dots, and the electron transfer is terminated, causing the phosphorescence of Mn-doped ZnS quantum dots recovery. Various factors including pH effect, the reaction time, the ion strength and the concentration of lysozyme-aptamer were optimized. The results showed that the liner range of the detection of lysozyme in our presentedsensing system was 5.50 nM-44.5 nM (R2 = 0.998) and the detection limit was 0.54 nM. To test the potential practical application of this system, we used the presented sensing system to determine the content of lysozyme in human serum and urine samples and the recoveries were found in the range of 104.5%–111.7% and 101.5%–102.7%, respectively. Therefore, our phosphorescence sensing system constructed a promising system which was simple, rapid, sensitive and selective for lysozyme detection and also exhibited potential application in biomedical field. [ABSTRACT FROM AUTHOR]