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Purpose: This study aimed to investigate the applicability of plasma extracellular vesicles (EVs) miR-99a-5p as a potential head and neck squamous cell carcinoma (HNSCC) diagnostic biomarker. Methods: The miRNA expression of HNSCC tissue and plasma EVs were profiled by small RNA sequencing. qRT-PCR was performed to detect miR-99a-5p expression in HNSCC ( n = 93) and benign disease ( n = 39) plasma EVs and formalin-fixed and paraffin-embedded (FFPE) tissue ( n = 110). We constructed receiver-operating characteristic curves to investigate the diagnostic efficiency of plasma EVs miR-99a-5p. Results: Tumor tissue exhibited lower miR-99a-5p than para-tumor tissue. Patients with high miR-99a-5p expression exhibited significantly more p16 positive status. In contrast, HNSCC plasma EVs harbored more miR-99a-5p than the benign disease group. Plasma EVs miR-99a-5p distinguished HNSCC with area under the curve (AUC) of 0.7494 (95% CI: 0.6692-0.8296; p < 0.0001), with 61.54% sensitivity and 75.27% specificity, respectively. Furthermore, plasma EVs miR-99a-5p also distinguished early HNSCC with AUC of 0.7394 (95% CI: 0.6284-0.8504; p = 0.0002), with 79.07% sensitivity and 61.54% specificity, respectively. Conclusion: Plasma EVs miR-99a-5p is a potential biomarker for predicting early HNSCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Huang, Shen, Hsueh, Zhang, Yuan, Zhou, Li, Lin, Wu and Hu.)