A method of detecting vitamin D in blood using laser desorption/ionization mass spectrometry (LDI-MS) and an apparatus therefor according to the present invention are not complicated in a measurement, do not require a number of measurement steps, and allow for easy measurement and collection of results in real time with a quick analysis. In addition, precise analysis may be performed even at a lower concentration of a sample, such that sensitivity and precision are excellent, various subtypes of vitamin D may be simultaneously detected, a throughput is high, and structural analysis and quantitative analysis of vitamin D that has undergone a metabolic process in blood may be accurately performed without a matrix interference.