To establish a method of determining melatonin in Cordyceps sinensis mycelium by high performance liquid chromatograhy electrospray inonization tendem mass spectrometry(HPLC-MS/MS).Methods Melatonin in the sample was extracted utrasonically with methanol. The content was then seperated on Thermo Beta Basic-18column (150mm×4.6mm, 5μm) with methanol/ 0.1% formic acid solution (60∶40) as mobile phase. The flow rate was 0.6ml/min, and column temperature was set to 40℃. Detection was performed under positive ion electrospray ionization tandem ion trap mass spectrometry (ESI+)-tandem mass spectrometry (MS/MS) mode.Results There was a good linearity of melatonin in the range of 10～200μg/kg with correlation coefficient of 0.9992. The detection limit of the method was 0.5μg/kg, and the limit of the quantification was 2.0μg/kg. The averange recovery was 81.92%. The relative standard deviration was lower than 10%.Conclusion This method is selective and fast. It can be applied to detect the melatonin in Cordyceps sinensis mycelium powder, and it may also provide basis for the determination in other complex systems.