Abstract: Droplet-based microfluidics is a technology that generates and manipulates highly uniform droplets, ranging from picoliter to nanoliter droplets, in microchannels under precise control. In biological research, each droplet can be used to encapsulate a small group of cells or even a single cell, and then serve as an individual container for biochemical reaction, which is well suited for high-throughput and high-resolution biochemical analysis. In the field of microbial research, from cultivation and identification of microbes to the investigation of the spatiotemporal dynamics of microbial communities, from precise quantitation of microbiota to systematic study of microbial interactions, and from the isolation of rare and unculturable microbes to the development of genetically engineered strains, droplet microfluidic technology has played an important promotional role in all these aspects. Droplet microfluidics shows potential for becoming a basic tool for exploring single-cell microbes in microbiological research. In this review, we gave a brief overview of the technical basis of droplet microfluidics. Then, we presented its latest applications in microbial research and had some discussions, aiming to provide a reference for relevant research on microorganisms.