The dideoxy chain-termination method, which was invented in 1977 by Sanger, has opened the door of DNA sequencing. The sequencing technology has continuously advanced during the past 30 years. Each new technology has unique advantages over the previous generation, but it also has its own limitations. The key is to recognize well both the advantages and disadvantages of each technology so that it can be used reasonably. The third generation sequencing technology is a new technology with many advantages, such as high throughput, fast speed, long read and low cost. Additionally, its emergence has advanced the study of genomics, transcriptomics, epigenomics and so on. In this review, the principle of the third generation sequencing technology is introduced, followed by a summary of its application to microbial research, and its potential uses in other areas.