The invention provides a substrate, a preparation method thereof and a mass spectrometric analysis detection method for metabolic molecules, and belong to the field of material synthesis and mass spectrometry. A zinc oxide substrate is synthesized by a hydrothermal method with sodium hydroxide and zinc acetate as raw materials, conditions are easy to control, the time consumed is short, and the operation is simple. The prepared zinc oxide substrate is star-shaped nanoparticles assembled by sword-shaped nanorods with sizes of less than 2 microns, particle sizes are uniform, and the substrate has good ultraviolet absorption properties, thereby having good properties being helpful of ionization analysis the a sample molecular. Compared with a spherical nano-zinc oxide substrate, the star-shaped zinc oxide substrate has smaller background noise, can remove the background interference and hot spot effect of traditional substrates, meanwhile, has better salt-tolerant and protein-resistant properties, and can be used for complex biological sample systems such as serums and the like. Simultaneously, sample pretreatment operations such as enrichment, separation and the like are not requiredin the detection process, and the efficient and rapid detection of the metabolic molecules is achieved.