With the rapid advancement of next-generation sequencing ( NGS ), sequencing and its related technologies are becoming more mature and providing higher accuracy and throughput. Traditional method of sequencing require a large sample quantity that is difficult to obtain for some samples. Thus, techniques with a lower demand on sample quantity are in urgent need. In library construction, the Tn5 transposase cuts DNA and adds an adapter sequence to both sides of the target sequence, which requires fewer steps and a lower sample quantity. Therefore, applications of Tn5 transposase have been gradually developed in biotechnology. For example, CUT&Tag, stLFR, and TRACE-seq are techniques that use Tn5 transposase in epigenomic sequencing, long fragment reading, and transcriptome sequencing. The simple steps and very low cost make the techniques popular. In this review, we summarize the Tn5 transposon and its applications in biotechnology to make it understood more easily and provide a reference for improvement and promotion in the future.