AbstracttRNAs are a group of conventional noncoding RNAs (ncRNAs) with critical roles in the biological synthesis of proteins. Recently, tRNA-derived small RNAs (tsRNAs) were found to have important biological functions in the development of human diseases including carcinomas, rather than just being considered pure degradation material. tsRNAs not only are abnormally expressed in the cancer tissues and serum of cancer patients, but also have been suggested to regulate various vital cancer hallmarks. On the other hand, the application of tsRNAs as biomarkers and therapeutic targets is promising. In this review, we focused on the basic characteristics of tsRNAs, and their biological functions known thus far, and explored the regulatory roles of tsRNAs in cancer hallmarks including proliferation, apoptosis, metastasis, tumor microenvironment, drug resistance, cancer stem cell phenotype, and cancer cell metabolism. In addition, we also discussed the research progress on the application of tsRNAs as tumor biomarkers and therapeutic targets.