One of the most common gastrointestinal tumors is gastric cancer (GC), which has a high lethality and a poor prognosis. Recently, it was discovered that mature tRNAs, which are expressed differently in a variety of malignancies, give rise to a novel class of tRNA-derived small RNAs (tsRNAs). In this study, we investigated the role of short RNAs produced from tRNA in GC and possible therapeutic uses. edgeR was used to screen the differentially expressed tsRNAs from the TCGA database and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was used to verify the levels of tsRNAs in GC samples. tRF-24-6VR8K09LE9 downregulated in GC was confirmed by detecting serum samples from 114 patients with gastric cancer, 40 patients with gastritis, and 100 healthy controls. The chi-square test displayed that tRF-24-6VR8K09LE9 was highly related to differentiation grade (P = 0.029), T-stage (P = 0.036), lymph node status (P = 0.036), TNM staging (P <
0.0001), and neurological/vascular invasion (P = 0.033). The receiver operating characteristic (ROC) curve indicated that tRF-24-6VR8K09LE9 is more effective than the current diagnostic markers for GC. Furthermore, mechanistic studies verified that tRF-24-6VR8K09LE9 affected the malignant progression of GC through the PI3K/AKT signaling pathway. In conclusion, tRF-24-6VR8K09LE9 can be served as a molecular marker for early GC auxiliary diagnosis. Over-expression of tRF-24-6VR8K09LE9 inhibits the malignant progression of GC, which may provide a new strategy for the adjuvant treatment of GC.