tRNA-derived fragments (tRFs) are a new category of regulatory noncoding RNAs with distinct biological functions in cancers and stress-induced diseases. Herein, we first summarize the classification and biogenesis of tRFs. tRFs are produced from pre-tRNAs or mature tRNAs. Based on the incision loci, tRFs are classified into several types: tRF-1, tRF-2, tRF-3, tRF-5, and i-tRF. Some tRFs participate in posttranscriptional regulation through microRNA-like actions or by displacing RNA binding proteins and regulating protein translation by promoting ribosome biogenesis or interfering with translation initiation. Other tRFs prevent cell apoptosis by binding to cytochrome c or promoting virus replication. More importantly, the dysregulation of tRFs has important clinical implications. They are potential diagnostic and prognostic biomarkers of gastric cancer, liver cancer, breast cancer, prostate cancer, and chronic lymphocytic leukemia. tRFs may become new therapeutic targets for the treatment of diseases such as hepatocellular carcinoma and respiratory syncytial virus infection. Finally, we point out the existing problems and future research directions associated with tRFs. In conclusion, the current progress in the research of tRFs reveals that they have important clinical implications and may constitute novel molecular therapeutic targets for modulating pathological processes.