High quality PEI-capped Mn-doped ZnS QDs were prepared via a microwave and ultrasonic-assisted synthesis for room temperature phosphorescence detection of guanosine 5’-triphosphate(GTP).Such QDs showed high selectivity and sensitivity for GTP，and could distinguish GTP from adenosine triphosphate(ATP)，cytidine triphosphate(CTP)，uridine triphosphate(UTP)，adenosine diphosphate(ADP) and adenosine monophosphate(AMP).The room temperature phosphorescence response of the PEI-capped Mn-doped ZnS QDs for GTP reached equilibrium in 3 min.PEI-capped Mn-doped ZnS QDs gave a detection limit(3s) of 0.6 μmol/L for GTP.The developed phosphorescence probe favors biological applications since the interference from scattering light and autofluorescence can be effectively eliminated.The developed method has been successfully applied for the room temperature phosphorescence detection of GTP in cancer cell extract.