Abstract: With the rapid development of modern detection technology and scientific research, the chemical structures and functions of isomers are constantly being revealed. The isomer molecules may have completely different biochemical activities, and a certain molecule of the isomer may play an important role in disease pathogenesis or disease treatment, while the other is the opposite. Therefore, the separation and identification of isomers and the exploration of their structures are helpful to furtherly understand the operating mechanism of life, and become one of the research hotspots in the field of analytical chemistry and life sciences. Ion mobility spectrometry (IMS), which has developed rapidly in recent years, is an instrument technology that can analyze the spatial structure of molecules. It can distinguish the isomerism of a certain molecule by measuring the difference in the collision cross-section between different molecular ions and airflow in the gas phase. IMS coupled with mass spectrometry (IMS-MS) is a powerful separation and analysis technology that adds a mobility separation dimension for MS to improve ion structure separation and determination, while also enhances ion mass selection in mobility technology and makes the separation of the analyte have high selectivity. IMS-MS technology has been highly concerned and widely used in the fields of small molecule/metabolite identification, proteomics research, host-guest complexes, and molecular structure identification. This paper was focused on the working principle of different types of IMS, the latest application in different types of isomers and the prospect of IMS-MS.