Deep analysis of next-generation sequencing data unveils numerous small non-coding RNAs with distinct functions. Recently, fragments derived from tRNA, named as tRNA-derived small RNA (tsRNA), have attracted broad attention. There are mainly two types of tsRNAs, including tRNA-derived stress-induced RNA (tiRNA) and tRNA-derived fragment (tRF), which differ in the cleavage position of the precursor or mature tRNA transcript. Emerging evidence has shown that tsRNAs are not merely tRNA degradation debris but have been recognized to play regulatory roles in many specific physiological and pathological processes. In this review, we summarize the biogeneses of various tsRNAs, present the emerging concepts regarding functions and mechanisms of action of tsRNAs, highlight the potential application of tsRNAs in human diseases, and put forward the current problems and future research directions.