Objective:To detect the changes in the levels of miR-21-5p and miR-125a-5p modified with 3′-terminal 2′-O-methylation (3′t-2′Ome) in serum extracellular vesicles (EV) of non-small cell lung cancer (NSCLC) patients, and evaluate their value as auxiliary screening molecular markers for NSCLC patients.Method:A retrospective analysis was conducted on the data of 69 NSCLC patients diagnosed at the Eastern Theater Command General Hospital from May 1st to October 31st,2023, as well as 65 age and gender matched healthy controls during the same period. Two real-time fluorescence quantitative PCR (RT-qPCR) techniques, namely stem-loop method and poly (A) tailed method, were used to detect the levels of 3′t-2′Ome-miR-21-5p and 3′t-2′-Ome miR-125a-5p in serum EV of NSCLC patients and controls.Analyze the correlation between the levels of two types of 3′t-2 ′Ome miRNAs and the differences in clinical stage, pathological classification, and other tumor indicators in patients. Receiver operating characteristic (receiveroperating curve, ROC) curves were used to analyze the efficacy of 3′t-2 ′Ome miR-21-5p and 3′t-2′ Ome miR-125a-5p in serum EV, as well as their combination, in diagnosing NSCLC.Result:Compared with the control group, the levels of 3′t-2′Ome-miR-21-5p in serum EV of NSCLC patients increased [(0.30±0.05) vs (0.35±0.09), t=3.32, P=0.001], while the levels of 3′t-2′Ome-miR-125a-5p decreased [(0.33±0.06 vs 0.25±0.06, t=7.45, P<0.001]. The differences were statistically significant. The expression levels of 2′Ome-miR-21-5p in EV were also significantly elevated in the NSCLC patients at 0-Ⅱ stage, adenocarcinoma patients, and squamous cell carcinoma patients, respectively. Notably, the levels of EV 3′t-2′Ome-miR-21-5p was also statistically significant between the adenocarcinoma patients and squamous cell carcinoma patients [(0.34±0.85) vs (0.40±0.12), P<0.05]. ROC curve analysis showed that the levels of 3′t-2 ′Ome miR-21-5p and 3′t-2′ Ome miR-125a-5p in serum EV, as well as their combined AUC for discriminating NSCLC patients, were 0.647(95% CI 0.550-0.743), 0.825(95% CI 0.756-0.894) and 0.860(95% CI 0.797-0.923), respectively. The sensitivity was 92.3%, 80.0%, 89.2%, and the specificity was 46.4%, 73.9%, and 78.3%, respectively. Conclusion:There are changes in the levels of 2 ′Ome miR-21-5p and 2′ Ome miR-125a-5p in the serum EV of NSCLC patients, and the combined detaction has the potential as an auxiliary screening molecular marker of NSCLC patients.