The invention relates to a method for detecting diphenoxylate in a biological sample by liquid chromatography-tandem mass spectrometry for criminal investigation. The method comprises the following steps: (I) extracting diphenoxylate from the biological sample; and (II) detecting diphenoxylate extracted in the step (I) by a liquid chromatography-tandem mass spectrometry instrument. The method provided by the invention improves the sensitivity of the detection of diphenoxylate in the biological sample to be detected, and reduces the degree of detection difficulty of diphenoxylate in the biological sample to be detected.