The invention provides a method for detecting apis mellifera and apis cerana alpha-amylase in honey through liquid chromatography-tandem mass spectrometry. The method comprises the steps: screening characteristic peptide fragments belonging to apis mellifera and apis cerana alpha-amylase respectively, establishing a liquid chromatography-tandem mass spectrometry detection method, pretreating a sample, carrying out liquid chromatography separation and tandem mass spectrometry detection, carrying out data analysis and the like, wherein the characteristic peptide fragment comprises an apis mellifera alpha-amylase characteristic peptide fragment VCLPPGQYCDVISGNLEK and an apis cerana alpha-amylase characteristic peptide fragment LVDFLDLVAVGVAGFR. The detection method provided by the invention has high accuracy, precision and sensitivity and strong stability, is suitable for identification of apis mellifera and apis cerana, and is especially suitable for identification of apis mellifera doped in apis cerana. The method has important practical significance for protecting rights and interests of honey consumers and maintaining healthy development of the bee product industry.