Abstract: The endogenous small molecule metabolites (such as neurotransmitters, fatty acids, carbohydrates, nucleotides, and amino acids) in the body played an important role in the development and progression of central nervous system diseases. With the emergence of precision medicine, an increasing demand for the precise localization and precise analysis of those metabolites, as well as a clearer understanding of their biological functions was presented. Mass spectrometry imaging (MSI) technology, a non-invasive in vivo imaging technique, could provide an accurate spatial distribution, and the concentrations of endogenous metabolites or pharmaceutical components in different organs. There were many advantages including no specific labeling, short duration, and wide detection range. Matrix-assisted laser desorption/ionization mass spectrometry imaging played a leading role in the study of the precise distribution of endogenous metabolites in the brain microregions of disease animal model such as Parkinson's disease, Alzheimer's disease, stroke, and insomnia. In addition, it was widely applied to study the distribution of central nervous system drugs in the brain microregions. This review systematically examines the fundamental principles underlying MSI and its groundbreaking technological innovations over the past decade.