Environmental microbiology has been an essential part of environmental research because it provides effective solutions to most pollutants. Hence, there is an interest in investigating microorganism behavior, such as observation, identification, isolation of pollutant degraders, and interactions between microbial species. To comprehensively understand cell heterogeneity, diverse approaches at the single-cell level are demanded. Thus far, the traditional bulk biological tools such as petri dishes are technically challenging for single cells, which could mask the heterogeneity. Single-cell technologies can reveal complex and rare cell populations by detecting heterogeneity among individual cells, which offers advantages of higher resolution, higher throughput, more accurate analysis, etc. Here, we overviewed several single-cell techniques on observation, isolation, and identification from aspects of methods and applications. Microscopic observation, sequencing identification, flow cytometric identification and isolation, Raman spectroscopy-based identification and isolation, and their applications are mainly discussed. Further development on multi-technique integrations at the single-cell level may highly advance the research progress of environmental microbiology, thereby giving more indication in the environmental microbial ecology.