tRNA-derived small RNAs (tsRNAs) is a type of newly discovered non-coding small RNA, which is derived from mature tRNA or tRNA precursor and exists in various organisms. tsRNAs exhibit tissue- and cell-specific expression and are involved in various biological functions such as stress response, protein translation regulation, ribosomes biogenesis, intergenerational transmission of acquired epigenetic information, tumorgenesis, cell proliferation and apoptosis. This review briefly discussed the biogenesis, classification, biological functions and molecular mechanisms of tsRNAs, as well as the roles of tsRNA modifications in tsRNA regulation and in diseases diagnosis.