In recent years, next-generation sequencing (NGS) technologies targeting the microRNA (miRNA) transcriptome revealed the existence of tRNA-derived short RNAs: tRNA halves (tiRNAs) and tRNA-derived fragments (tRFs). These small RNAs represent a novel type of small non-coding RNAs (sncRNAs), which are heterogeneous in size, nucleotide composition and biogenesis, and have been suggested to be involved in translation, cell proliferation, priming of viral reverse transcriptases, regulation of gene expression, modulation of the DNA damage response, tumor suppression and neurological disorders. Herein, we review the mechanism of their biogenesis and discuss in detail the regulatory roles they play in cell physiology. We also point out that the biological function of tRNA-derived short RNAs will be understood better as research moves forward, and that this knowledge will find its way into clinical application in the near future.