BACKGROUND: The development of biomarkers for the early detection of non-small cell lung cancer (NSCLC) is clinically important. We have developed miRNA biomarkers in sputum and plasma, respectively, for NSCLC. Herein, we evaluate whether integrated analysis of the miRNAs across the different types of specimens could improve the early detection of NSCLC. METHODS: Using reverse transcription PCR, we determined expressions of two miRNAs (miRs-31-5p and 210-3p) in sputum and three miRNAs (miRs-21-5p, 210-3p, and 486-5p) in plasma of a training cohort of 76 NSCLC patients and 72 cancer-free smokers. The results were validated in a testing cohort of 56 NSCLC patients and 55 cancer-free smokers. RESULTS: The panels of two sputum miRNAs and three plasma miRNAs had 65.8-75.0% sensitivities and 83.3-87.5% specificities for diagnosis of NSCLC in the training cohort. The individual sputum or plasma miRNA panel had a higher sensitivity for squamous cell carcinoma or adenocarcinoma of the lung, respectively. From the miRNAs, we optimized an integrated panel of biomarkers consisting of two sputum miRNAs (miRs-31-5p and 210-3p) and one plasma miRNA (miR-21-5p) that had higher sensitivity (85.5%) and specificity (91.7%) for diagnosis of NSCLC compared with the individual panels alone. Furthermore, the performance of the integrated panel of biomarkers was independent of histology and stage of NSCLC, and patients' age, sex, and ethnicity. The performance of the integrated panel of biomarkers was confirmed in the testing cohort. CONCLUSIONS: Integrating biomarkers across different body fluids would synergistically improve the early detection of NSCLC. KEY POINTS: Lung cancer is a heterogeneous disease and develops from complex aberrations. Integrating sputum and plasma miRNAs has higher accuracy than when they are used alone.