Exosome-associated tRNA-derived fragments (tRFs) play crucial roles in cancer progression, influencing cell proliferation, metastasis, immune evasion, and drug resistance. Due to their stability and specific expression in bodily fluids, exosomal tRFs hold great promise as non-invasive biomarkers for early cancer diagnosis, treatment monitoring, and prognosis evaluation. Additionally, tRFs present opportunities as therapeutic targets, with potential to modulate oncogenic processes, such as controlling gene expression and modulating cellular signaling pathways. This review explores the diverse functions of exosome-associated tRFs in tumor biology, highlighting their potential for clinical applications, including their use as diagnostic tools and their role in therapy. It also addresses the challenges that remain in standardizing detection methods and validating their efficacy in clinical settings, such as variability in isolation techniques and the need for large-scale studies. Advanced technologies and further research will be key to unlocking their potential in personalized cancer therapy, ultimately aiming to integrate tRFs into routine clinical practice for better patient outcomes.