Recent progress in noncoding RNA research has highlighted transfer RNA-derived small RNAs (tsRNAs) as key regulators of gene expression, linking them to numerous cellular functions. tsRNAs, which are produced by ribonucleases such as angiogenin and Dicer, are classified based on their size and cleavage positions. They play diverse regulatory roles at the transcriptional, post-transcriptional, and translational levels. Furthermore, tRNAs undergo various modifications that influence their biogenesis, stability, functionality, biochemical characteristics, and protein-binding affinity. tsRNAs, with their aberrant expression patterns and modifications, act as both oncogenes and tumor suppressors. This review explores the biogenetic pathways of tsRNAs and their complex roles in gene regulation. We then focus on the importance of RNA modifications in tsRNAs, evaluating their impact on the biogenesis and biological functions on tsRNAs. Furthermore, we summarize recent data indicating that tsRNAs exhibit varied expression profiles across different cancer types, highlighting their potential as innovative biomarkers and therapeutic targets. This discussion integrates both existing and new knowledge about tsRNAs, emphasizing their importance in cancer biology and clinical advancement.