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.