Objective: Our study aimed to investigate the expression level and clinical significance of serum and exhaled breath condensate miR-186 and IL-1β in non-small cell lung cancer patients. Methods: The serum and exhaled breath condensate specimens of 62 non-small cell lung cancer patients and 60 healthy controls were collected to detect miR-186 expression levels by real-time fluorescent quantitative PCR. Enzyme linked immunosorbent assay was applied to examine IL-1β concentration. Statistical analyses were used to evaluate the correlation between miR-186 and IL-1β in serum and clinicopathological features, traditional serum tumor markers, and inflammatory markers. The diagnostic efficacy of miR-186 and IL-1β for non-small cell lung cancer was evaluated by receiver operating characteristic curve analysis. The correlation between miR-186 and IL-1β was determined. Results: ① The relative expression level of miR-186 was greatly reduced in the serum and EBC of patients with non-small cell lung cancer, and the miR-186 expression level was reduced in different TNM stages of non-small cell lung cancer, from the early to later stages. ② The IL-1β concentration in serum and exhaled breath condensate of patients with non-small cell lung cancer was increased. ③ Serum miR-186 and IL-1β levels were closely related to lymph node metastasis, and the low expression of serum miR-186 and the high concentration of IL-1β were associated with higher serum carcinoembryonic antigen, C-reactive protein, and erythrocyte sedimentation rate levels. ④ ROC curve analysis showed that exhaled breath condensate miR-186 had higher area under the curve than serum miR-186, and the combined detection showed higher diagnostic efficacy than the separate detection. In addition, the combined detection of IL-1β and miR-186 has a larger AUC than the separate detection of both. ⑤ The correlation between serum miR-186 and IL-1β was negative. Conclusion: miR-186 and IL-1β are expected to be potential diagnostic biomarkers for non-small cell lung cancer.