The discovery of the DNA double helix structure has turned life science research into the molecular level, and the sequencing technology that emerged in the 1970s has made a great contribution to the deciphering of the genetic code. Single-molecule sequencing technology, also known as third-generation sequencing technology, which has appeared in recent years, can read nucleotide sequences at the single molecular level. The single-molecule sequencing systems mainly include HeliScope, Nanopore and PacBio. Compared to traditional first- and next-generation sequencing technologies, third-generation sequencing can produce longer reads, sequence RNA directly without reverse transcription, and the speed is extremely fast. Meanwhile the equipment of some systems can be miniaturized and portable for in-field sequencing. The third-generation sequencing technology has numerous applications in the basic theroretical research of life science and the clinical practice in biomedicine. This paper focuses on the principles, the pros and cons, and the research progress and applications, of various single-molecule sequencing methods.