In 1975, the sequencing of the first cDNA genome, the phage фX174 genome, marked the beginning of the sequencing era. Since 1996, the launch of the Human Genome Project has greatly promoted the application and development of sequencing technology. The second generation sequencing technology is also known as high-throughput sequencing technology. Single-molecule DNA sequencing technology is a new generation of sequencing technology called third-generation sequencing technology, which developed in the last 10 years. It includes single-molecule real-time sequencing, true single-molecule sequencing and single-molecule nanopore sequencing. Because SMRT sequencing technology has the characteristics of long reading length, short sequencing period, without requirement for template amplification and direct detection of apparent modification sites, it provides researchers with new options. This paper reviews the basic principle and performance of SMRT sequencing technology and its application in microbial 16S rRNA gene, microbial whole genome sequencing and microbial macrogenome sequencing, and analyzes the advantages and problems of SMRT sequencing technology in environmental microorganisms.