Exosomes are extra-cellular vesicles that are < 150 nm that is formed by invagination of the plasma membrane and are released as vesicles. These contain proteins, RNA, and DNA as their cargo. In recent times, the non-coding RNA (ncRNA) present within exosomes has been studied extensively in the context of sorting, localization, and their potential as biomarkers. For example, miR-1246, miR-1290, miR-21, and miR-23a are exosomal biomarkers of cancer, and YBX1 (Y-Box Binding Protein 1) is attributed to exosomal RNA sorting. Transfer RNA-derived fragments are a class of small ncRNAs that were discovered in 2009. They are classified as tRFs (tRNA-derived fragments) and tsRNAs (tRNA halves). Interestingly, these tRNA-derived ncRNAs are emerging as biomarkers in various diseases, and these are found in exosomes. To date, the literature has covered only the biomarker potential of plasma/serum tRNA-derived ncRNAs. Hence, in the current review, we discuss the exosomal tRNA-derived fragments that are clinically relevant in pathological conditions.