Non-small cell lung cancer (NSCLC) is the first cause of cancer related death in the world. Biomarkers to predict the relapse and drug resistance could be extremely useful for a clinical doctor to monitor high risk patients and select rational regimen. miRNAs play an important role in lung cancer and detection samples are relatively easy to be obtained, miRNAs could become a promising means of comprehending the oncogenesis and pathogenesis of lung cancer. This study aimed to investigate the function of miR-145 to work as a biomarker in NSCLC. miR-145 expression level in 48 NSCLC tumor tissues and their matched normal tissues were detected by qRT-PCR. miR-145 in 18 paraffin-embedded samples underwent chemotherapy and were assessed by in situ hybridization (ISH). Here we show that miR-145 was down-regulated in NSCLC tissues; down-regulation of miR-145 was correlated with late clinical stage and poorly differentiated carcinoma, and, low expression level of miR-145 could also predict chemotherapy resistance and shorter disease-free survival (DFS). These findings indicated that miR-145 expression may be a useful prognostic marker that could be used for predicting poor differentiation, chemo-resistance and shore DFS.