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As our understanding of environmental microbial ecosystems deepened, the demand for in-depth studies in this area continued to grow. Traditional methods such as culturomics and environmental DNA sequencing were no longer sufficient to fully understand the species and functional diversity of microbes. The single-cell isolation and sequencing technology emerged as a new paradigm in environmental microbiology, representing a hot topic and cutting-edge advancement. This single-cell technology shifted the focus of microbial research from the community level to the individual cell level, allowing for the precise isolation and sequencing of single microbial cells. This approach provided a deeper understanding of the interspecies and intraspecies diversity and functional characteristics of microbes. This review introduced the application of single-cell technology in environmental microbiology, elaborated on the key processes of single-cell isolation and genome amplification, and discussed the bioinformatic flow of the single-cell sequencing data analysis. It aimed to provide insights and inspiration for the development of single-cell technology in environmental microbiology. [ABSTRACT FROM AUTHOR]