An unique subclass of functional non-coding RNAs generated by transfer RNA (tRNA) under stress circumstances is known as tRNA-derived small RNA (tsRNA). tsRNAs can be divided into tRNA halves and tRNA-derived fragments (tRFs) based on the different cleavage sites. Like microRNAs, tsRNAs can attach to Argonaute (AGO) proteins to target downstream mRNA in a base pairing manner, which plays a role in rRNA processing, gene silencing, protein expression and viral infection. Notably, tsRNAs can also directly bind to protein and exhibit functions in transcription, protein modification, gene expression, protein stabilization, and signaling pathways. tsRNAs can control the expression of tumor suppressor genes and participate in the initiation of cancer. It can also mediate the progression of diseases by regulating cell viability, migration ability, inflammatory factor content and autophagy ability. Precision medicine targeting tsRNAs and drug therapy of plant-derived tsRNAs are expected to be used in clinical practice. In addition, liquid biopsy technology based on tsRNAs indicates a new direction for the non-invasive diagnosis of diseases.