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tRNA-derived small RNAs (tsRNAs) are derived from tRNA and include tRNA halves (tiRNAs) and tRNA fragments (tRFs). tsRNAs have been implicated in a variety of important biological functions, such as cell growth, transcriptional regulation, and apoptosis. Emerging evidence has shown that Ago1-guided and Ago2-guided tsRNAs are expressed at 3 and 30 days in <i>Drosophila</i> and that tRF biogenesis in fruit flies affects tRNA processing and tRNA methylation. However, a wide analysis of tsRNA patterns in different ages of <i>Drosophila</i> have not been reported via the small RNA sequencing method. In the present study, tsRNAs of young (7 days) and old (42 days) <i>Drosophila</i> were sequenced and their expression characteristics were analysed. Then, a specific tRF (named <i>tRF-Trp-CCA-014</i>) was determined and was found to be conserved in fruit flies, mice, and humans. The expression patterns of <i>tRF-Trp-CCA-014</i> in different tissues and stages of fruit flies and mice, and mouse NIH/3T3 cells were detected. Furthermore, mouse embryonic fibroblast NIH/3T3 cells were used as a model to analyse the function and targets of <i>tRF-Trp-CCA-014</i>. The RNA-seq data of six groups (Mimics, Mimic NC, Inhibitors, Inhibitor NC, Aging (adriamycin), and Control (Normal)) in mouse NIH3T3 cells were analysed. The results showed that the number of tsRNAs at 42 days (417) was more than at 7 days (288); thus, it was enriched with age. tRFs-1 were the most enriched, followed by 5′-tRFs and 3′-tRFs. Twenty-one differentially expressed tsRNAs were identified between 7 days and 42 days. Then, the conserved tRF <i>tRF-Trp-CCA-014</i> was identified and found to accumulate in aged fruit flies and aged mouse NIH3T3 cells. RNA-seq data showed that most differentially expressed genes were involved in the immune system, cancer: overview, and signal translation. Furthermore, <i>tRF-Trp-CCA-014</i> was found to bind to the 3′UTR of <i>H3C4</i> in a dual-luciferase reporter gene assay. <i>tRF-Trp-CCA-014</i> and <i>H3C4</i> were detected in the cytoplasm of aged NIH3T3 cells by RNA in situ hybridization. These results suggest that the <i>H3C4</i> gene is the target of <i>tRF-Trp-CCA-014</i>. This study will advance the current understanding of tRF roles and their implication in <i>Drosophila</i> and mouse studies.