Background: Extracellular vesicles (EVs) play important roles in intercellular communication through the delivery of their cargoes, which include proteins, lipids, and RNAs. Increasingly, multiple studies have reported the association between EV small non-coding RNAs and cancer, due to their regulatory functions in gene expression. Hence, analysis of the features of small non-coding RNA expression and their incorporation into EVs is important for cancer research. Results: We performed deep sequencing to investigate the expression of small RNAs in plasma EVs from lung adenocarcinoma (ADC) patients, lung squamous cell carcinoma (SQCC) patients, and healthy controls. Then, eighteen differently expressed miRNAs in plasma EVs was validated by QRT-PCR. The small RNA expression profiles of plasma EVs were different among lung ADC, SQCC patients, and healthy controls. And many small RNAs, including 5' YRNA hY4-derived fragments, miR-451a, miR-122-5p, miR-20a-5p, miR-20b-5p, miR-30b-5p, and miR-665, were significantly upregulated in non-small cell lung cancer (NSCLC) EVs. And the cell viability assays indicated that hY4-derived fragments inhibited the proliferation of lung cancer cell A549. By comparing the cellular and EV expression levels of six miRNAs in NSCLC cells, we found that miR-451a and miR-122-5p were significantly downregulated in NSCLC cell lysates, while significantly upregulated in NSCLC EVs. Conclusions: The differently expressed EV small RNAs may serve as potential circulating biomarkers for the diagnosis of NSCLC. Particularly, YRNA hY4-derived fragments can serve as a novel class of biomarkers, which function as tumor suppressors in NSCLC. Additionally, miR-451a and miR-122-5p may be sorted into NSCLC EVs in a selective manner.