tRNA-derived small RNAs (tsRNAs) are non-coding small RNAs produced by specific endonucleases following the processing and splicing of precursor or mature tRNAs upon starvation, oxidative stress, hypoxia, and other adverse conditions. tRNAs are classified into two major categories, tRNA fragments (tRFs) and tRNA-derived stress-induced small RNAs (tiRNAs), based on differences in splice sites. With the development of high-throughput sequencing technologies in recent years, tsRNAs have been found to have important biological functions, including inhibition of apoptosis, epigenetic regulation, cell–cell communication, translation, and regulation of gene expression. Additionally, these molecules have been found to be aberrantly expressed in various diseases and to be involved in several pathological processes. In this article, the classification and nomenclature, biological functions, and potential use of tsRNAs as diagnostic biomarkers and therapeutic targets in non-neoplastic diseases are reviewed. Although tsRNA research is at its infancy, their potential in the treatment of non-tumor diseases warrants further investigation.