An ultra high performance liquid chromatography coupled with a triple quadrupole mass detection and electrospray ionization mass spectrometry method has been established for the simultaneous determination of the 14 nucleosides and nucleobases, 24 amino acids and two main alkaloids in mulberry leaf. In this method, a complicated mobile phase, the flow rate of which was 0.4 mL/min, was applied to the gradient elution, which provided a satisfied separation of the 40 compounds. The present method was validated, and sufficient reproducibility and accuracy were obtained for the quantitative measurement of the 40 compounds. The method was subsequently applied to ten mulberry leaves and the results showed that almost all of these samples were rich in nucleosides, nucleobases, amino acids, and alkaloids. The proposed method, which is convenient and economical, could serve as a prerequisite for the quality control of mulberry leaf herbs and be applied analogously to other Chinese medicines.