The invention relates to the field of analytical chemistry, and discloses a liquid chromatography-mass spectrometry method for detection of biotin, folic acid and vitamin B12. The detection method uses alkaline water and ultrasonic extraction for the pretreatment of the sample to be tested, uses high performance liquid chromatography-mass spectrometry technology for the determination of appropriate conditions, so as to achieve the simultaneous detection of biotin, vitamin B12 and folic acid. The detection method has the advantages of high accuracy, precision and sensitivity, reduced test cost, and improved test efficiency.