BACKGROUND: tRNA halves (tiRNAs) are produced from mature tRNAs. They have important roles both with in normal cells and cancer cells. However, the diagnostic value of tiRNAs in cancers have not yet been elucidated. OBJECTIVE: To explore the diagnostic value of tiRNA-5034-GluTTC-2 in gastric cancer. PATIENTS AND METHODS: Quantitative reverse transcription-polymerase chain reaction was used to detect the expression levels of tiRNA-5034-GluTTC-2 in paired gastric cancer tissues and adjacent normal tissues, plasmas from patients with gastric cancer and healthy people, and gastric cancer cell lines. Then, the relationship between its levels and clinicopathological factors of patients with gastric cancer was analyzed. A receiver operating characteristic (ROC) curve was established to predict the diagnostic value. RESULTS: tiRNA-5034-GluTTC-2 was first found to be down-regulated in gastric cancer tissues and plasmas. Its levels were significantly associated with tumor size. The area under the ROC curve (AUC) was 0.779 and 0.835 in tissue and plasma, respectively. The sensitivity, specificity and AUC were 84.7%, 92.8%, and 0.915 when tissues and plasmas were used in combination, respectively. The overall survival rate of patients with a lower expression of tiRNA-5034-GluTTC-2 was significantly lower than those with a higher expression. CONCLUSIONS: These results indicated that tiRNA-5034-GluTTC-2 may be a novel biomarker for the diagnosis of gastric cancer.