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Non-small cell lung cancer (NSCLC) is a malignant tumor. Serum exosomal miR-27b is related to tumor diagnosis. We explored the roles of serum exosomal miR27b in NSCLC. NSCLC patients were assigned to NSCLCearly/terminal groups, with healthy subjects as controls. miR-27b expression was assessed using reverse transcription-quantitative polymerase chain reaction, and its diagnostic efficiency was analyzed using the receiver operating characteristic curve. The correlation between serum exosomal miR-27b expression and tumor markers carcinoembryonic antigen 125 (CA125), carcinoembryonic antigen (CEA), and cytokeratin 19-soluble fragment (CYFRA21-1) was analyzed using the Pearson analysis. The downstream target genes were predicted. Epidermal growth factor receptor (EGFR)level was assessed using enzyme-linked immunosorbent assay. Correlations of miR-27b expression with serum EGFR level and CA125, CEA, and CYFRA21-1 levels were analyzed using the Pearson analysis. Serum exosomal miR-27b was diminished in NSCLC and was further decreased in the NSCLS-terminal group. The sensitivity of miR-27b < 0.8150 for NSCLC diagnosis was 76.64%, and the specificity was 83.33%. Serum exosomal miR-27b was negatively correlated with CA125, CEA, and CYFRA21-1. miR-27b targeted EGFR. Serum EGFR was raised in NSCLC and was further elevated in the NSCLS-terminal group. miR-27b expression was negatively correlated with EGFR level. EGFR level was positively correlated with CA125, CEA, and CYFRA21-1 levels. Collectively, low expression of miR-27b assisted [ABSTRACT FROM AUTHOR]