Transfer RNAs (tRNAs) were traditionally considered to participate in protein translation. Recent studies have identified a novel class of small non-coding RNAs (sncRNAs), produced by the specific cleavage of pre- and mature tRNAs, which have been named tRNA-derived fragments. tRNA-derived fragments are classified into diverse subtypes based on the different cleavage positions of the pre- and mature tRNAs. Recently, accumulated evidence has shown that these tRNA-derived fragments are frequently dysregulated in several cancers. Several tRNA-derived fragments were found to participate in cell proliferation, apoptosis, and invasive metastasis in several malignant human tumors. These dysregulated fragments are able to bind both Argonaute proteins and Piwi proteins to regulate gene expression. Some of the newly identified tRNA-derived fragments have been considered as the new biomarkers and therapeutic targets for the treatment of cancer. This review summarizes the biogenesis and biological functions of different subtypes of tRNA-derived fragments and discusses their molecular mechanisms in cancer progression.