<p>Early diagnosis is vitally important for effective treatment of nasopharyngeal carcinoma (NPC). Nevertheless, the exact pathogenic mechanisms of NPC remain unclear, and early diagnosis of NPC is still limited. Herein, we showed that a specific tsRNA for NPC, 5′tiRNA-32-ValAAC-2, is a novel pathogenic factor and has potential diagnostic value for NPC screening. In this study, small RNA microarray profiling and array hybridization were used to detect expression spectrums of tsRNAs in the sera of newly diagnosed NPC patients. The upregulated tsRNAs were validated using RT-qPCR, and their clinical significance in NPC diagnosis was analyzed. Furthermore, the most highly expressed tsRNA, was further investigated. 5′tiRNA-32-ValAAC-2 could serve as a potential diagnostic biomarker for NPC. Subsequently, the effect of 5′tiRNA-32-ValAAC-2 on the growth and invasion of NPC cells was investigated. The results indicated that overexpression of 5′tiRNA-32-ValAAC-2 promoted NPC cells proliferation, migration, and invasion. In contrast, the inhibition of 5′tiRNA-32-ValAAC-2 suppressed NPC cells proliferation, migration and invasion. TargetScan and miRanda analyses revealed that <i>UGT2B7, SYNPO2, ZNF44, PDHB</i>, and <i>UFM1</i> might serve as downstream target-genes of 5′tiRNA-32-ValAAC-2. In conclusion, 5′tiRNA-32-ValAAC-2 could potentially be a novel pathogenic factor for NPC, and it functions as a diagnostic biomarker in the primary diagnosis of NPC.</p>