The invention discloses a high performance liquid chromatography tandem mass spectrometry detection method of 25-hydroxy vitamin D in serum. The method comprises the following steps that (1) a sampleis pre-treated, specifically, an internal standard working solution and a sodium hydroxide solution are sequentially added into a serum sample and mixed to be uniform; (2) solid-liquid extraction treatment is conducted, specifically, the sample treated in the step (1) is loaded to an SLE 96 pore plate, and eluted with normal hexane, and then the eluant is blown to be dried by using nitrogen; (3) aderivatization reaction is conducted, a derivatization reagent 4-phenyl-1,2,4-triazoline-3,5-diketone (PTAD) solution is added to redissolve, a thermostatic reaction is conducted, then ethyl alcoholis added to terminate the reaction, and the product is blown to be dried by using nitrogen; and (4) redissolution is conducted, and analytical detection is conducted by adopting the high performance liquid chromatography tandem mass spectrometry based on a multiplexing system (MPX). The high performance liquid chromatography tandem mass spectrometry detection method is high in detection sensitivity, high in specificity and low in cost, meanwhile, the pre-treatment time and analysis time of the sample are effectively shortened, and the detection efficiency of the sample is improved.