The invention discloses a high-performance liquid chromatography (HPLC)-tandem mass spectrometry detection method of 25-hydroxy vitamin D in dried blood spots. The method comprises the following steps: (1) ultrasonic extraction: taking a dried blood spots sample, adding an extraction solvent, ultrasonically extracting, and then adding an internal standard working solution, performing vortex-mixing, centrifuging, taking upper extraction liquid, drying through nitrogen; (2) derivatization reaction: adding the sample processed in the step (1) into a PTAD derivatization working solution to redissolve, reacting at constant temperature, and adding the ethanol to terminate the reaction, and drying through the nitrogen; and (3) redissolving: performing analysis detection by adopting the HPLC-tandem mass spectrometry. The method disclosed by the invention is high in efficiency, high in flux, low in cost, simple for operation, high in accuracy, and high in sensitivity, free form conflict limitation on other detection projects, and suitable for fast detecting batch of clinical samples; and meanwhile, the wound on the subject is small by adopting the dried blood spots method, the amount of collecting blood is less, the sample is convenient for collection and transportation, and easy to save, the stabiligy is good, and the method is especially suitable for the detection on the population (such as infants) difficult for venous blood collection.