The purpose of our research was to evaluate diagnostic performance of serum microRNA-135a (miR-135a) in non-small cell lung cancer (NSCLC).Quantitative real time-polymerase chain reaction was employed to detect the expression serum of miR-135a in NSCLC patients and controls. The influence of serum miR-135a level on clinical characteristics of NSCLC patients was explored through the Chisquare test. Serum carcinoembryonic antigen (CEA) level was estimated via enzyme-linked immunosorbent assay. Receiver operating characteristic (ROC) curve was plotted to elucidate diagnostic roles of serum miR-135a and CEA in NSCLC.The expression level of serum miR-135a was significantly lower in NSCLC patients than in healthy controls (0.40 ± 0.29 vs 1.00 ± 0.40, P < .001). Moreover, miR-135a expression was related to lymph node metastasis (P = .021), tumor differentiation (P = .020), and tumor node metastasis stage (P = .031). ROC curve showed serum miR-135a level could discriminate NSCLC patients from healthy controls (P < .0001) with a corresponding cutoff value of 0.665, and a sensitivity and specificity of 81.3% and 83.1%, respectively. The area under the curve was 0.888. In diagnosis analysis on the combination of miR-135a and CEA, when its specificity was maintained at 90%, diagnosis cut-off point reached 0.678.Serum miR-135a level is significantly downregulated in NSCLC and serves as a potential diagnostic biomarker for the disease.Abbreviations: AUC = area under the curve, CEA = carcinoembryonic antigen, ELISA = enzyme-linked immunosorbent assay, MicroRNA = miRNA, NSCLC = non-small cell lung cancer, QRT-PCR = quantitative real-time polymerase chain reaction, ROC = receiver operating characteristic, TNM = tumor node metastasis.