Abstract: Provided is a method for simultaneously detecting Vitamin K1 and Vitamin K2 in traces of blood. The method includes: constructing a two-dimensional liquid chromatography-tandem mass spectrometer, establishing an analytical method, and detecting at least three mixed standard solutions using the constructed two-dimensional liquid chromatography-tandem mass spectrometer to obtain a first detection result; fitting standard curve equations respectively corresponding to Vitamin K1 and Vitamin K2; and mixing and centrifuging a blood sample to which an extraction reagent and a certain amount of internal standard substance are added, collecting a supernatant, blowing the supernatant to dry with nitrogen, redissolving the residue, and detecting the dry supernatant using the constructed two-dimensional liquid chromatography-tandem mass spectrometer to obtain a second detection result. In this manner, concentrations of Vitamin K1 and Vitamin K2 in the blood sample are obtained.