The invention discloses a ferric oxide substrate, preparation and application of the ferric oxide substance in cerebrospinal fluid mass spectrometry. The ferric oxide substrate is a nano spherical particle, the size of the ferric oxide substrate is less than 1mu m, the particle size of the particle is uniform, and the nano spherical particle has a rough surface. The ferric oxide substrate can effectively eliminate background noise interference of a conventional organic substrate, so that the parsing ionization effect of small-molecule substances is greatly enhanced, and extremely high salt tolerance and protein tolerance are achieved. On the basis of the ferric oxide substrate, the invention provides a novel method for accurately quantifying molecules in a cerebrospinal fluid system, therefore, precise identification of the small-molecule substances is realized; and furthermore, the sample amount of cerebrospinal fluid can be reduced to a nano liter level, no organic substrate is added in the process, and no sample pretreatment is needed, so that efficient and quick detection of a low-molecular-weight compound is realized.