Electron microscopy (EM) is one of the most important techniques to characterize the morphology and structure of micro- and nano-particles. However, conventional procedures for sample preparation often alter the structure and morphology of samples with high water contents, causing distortions and misinterpretations for EM characterizations. The invention of Cryo-electron microscopy (Cryo-EM) has largely solved this problem. Via rapidly freezing the hydrated samples at low temperatures, cryogenic techniques instantaneously transform liquid water into amorphous ice to ensure high vacuum and reduce electron radiation damage, allowing researchers to observe hydrated samples in their native state with high resolution. For example, applications of Cryo-EM during the COVID-19 pandemic have demonstrated the amazing ability of Cryo-EM to visualize the detailed structure of SARS-CoV-2 viruses, which provides vital knowledge for rapid and reliable detection and diagnosis of the disease, transmission mitigation, and vaccine development. So far, Cryo-EM technology has been widely used in materials, biology, pharmaceuticals, and other fields of research, successfully broadening and deepening the understanding of the interactions between micro- and nano-particles. This review summarizes the recent development of Cryo-EM from aspects of electronic components, imaging technology, and resolution and introduces the sample preparation methods. Furthermore, the three-dimensional reconstruction method is highlighted to advance the EM method from 2D to 3D. As most environmental samples are highly hydrated, Cryo-EM will likely become an essential tool to investigate microscopic particles in the environmental field. This review gives examples of the applications of Cryo-EM in the formation of aerosol particles in the atmosphere, observing biofilm morphology in the water treatment process, the pore structure of activated sludge flocs, and the potential mechanism of soil microorganisms on heavy metal remediation. Finally, the prospects of Cryo-EM are summarized and discussed. We expect that with software, hardware, and artificial intelligence development, Cryo-EM technology can achieve faster data acquisition and higher resolution and make breakthrough contributions to environmental chemistry research.