Abstract: Mass spectrometry imaging technology is an emerging research field developed on the basis of mass spectrometry technology. It combines mass spectrometry technology with imaging software technology, which can visualize the molecules of the measured substances. This technique can be used to observe the spatial distribution of molecules in situ without special staining, contrast labeling and complex sample pretreatment. It has a good application prospect in the fields of plant tissue structure research, drug metabolism, clinical diagnosis, judicial detection and so on. The principle, operation process, sample processing, matrix selection and spraying of matrix-assisted laser desorption ionization mass spectrometry imaging were reviewed, and its applications in food analysis, pharmaceutical analysis, medical field and plant research in recent years were summarized as well.