Various studies have demonstrated the diagnostic value of microRNA (miRNA) for lung cancer, but miRNA signatures varied between different subtypes. Whether serum miRNAs could be used as biomarkers in lung squamous cell carcinoma (SCC) remains unknown. Using quantitative real-time polymerase chain reaction (qRT-PCR) based Exiqon panel, 38 differentially expressed miRNAs were identified from 3 male lung SCC pool samples and 1 normal control (NC) pool in the initial screening phase. After the training (24 SCC VS. 15 NCs), testing (44 SCC VS. 57 NCs) and external validation (34 SCC VS. 36 NCs VS. 10 pulmonary hamartoma) processes via qRT-PCR, we identified a three-miRNA panel ((miR-106a-5p, miR-20a-5p and miR-93-5p) to be a potential diagnostic marker for male lung SCC patients. The areas under the receiver operating characteristic (ROC) curve of the three-miRNA panel for the training, testing and validation phases were 0.969, 0.881 and 0.954 respectively. In addition, this signature could also differentiate lung SCC from pulmonary hamartoma (AUC=0.900). The 3 miRNAs were consistently up-regulated in lung SCC tissues (23 SCC VS. 23 NCs) and serum exosomes (17 SCC VS. 24 NCs). Moreover, expression of the 3 miRNAs was decreased in arterial serum (n = 3). In conclusion, we established a three-miRNA signature in the peripheral serum with considerable clinical value in the diagnosis of male lung SCC patients.