Raman microscopy is a technique that provides information on the chemical structure of materials within a spatial resolution of 0.5 to 1 μm. In recent years, Raman microspectroscopy, which can quickly and non-destructively detect characteristic chemical components in single cells, has been increasingly applied to microbiology. The Raman spectrum of a typical microbial cell contains information on nucleic acids, proteins, carbohydrates, lipids, and pigments (e.g. carotenoids) that characterize the genotype, phenotype, and physiological state of the microbial cells. Therefore, single-cell Raman microscopy is a “full bio-fingerprint” technique that can be used to distinguish different microbes. Raman microspectroscopy can be used to study the transformation of individual microbial cell life stages, identify changes in pigments and other compounds in single microbial cells. This paper reviews the current applications of Raman microspectroscopy on the study of microbiology at single cell level. Its combination with stable isotope probing (SIP), Raman imaging, spectral classification and cell sorting techniques that used to explore the microbial classifications and the relationships between genotypes and phenotypes, were also discussed in this paper. Collectively, Raman microspectroscopy is a promising method in the studies of cell sorting and metabolic analysis at single cell level.