Abstract: Objective To investigate the expression of miRNA-29s（miR-29s）and its clinical significance in non-small cell lung cancer（NSCLC）. Methods From January 2012 to December 2015，94 cases of surgical resected NSCLC tissues and 71 cases of adjacent normal tissues were collected. The levels of miR-29a，miR-29b and miR-29c in the above tissues were detected by fluorescence real-time quantitative PCR（QPCR）method. The miR-29s levels in NSCLC tissues and adjacent normal tissues were compared. The correlation among miR-29a，miR-29b and miR-29c were analyzed by Pearson correlation analysis. The relationship between miR-29s level and NSCLC clinicopathological parameters（gender，age，TNM stage，histological type，differentiation degree，lymph node metastasis and CEA level）were also analyzed. The relationship between different miR29s levels and prognosis were compared according to follow-up data. Results The QPCR results showed that the levels of miR-29a，miR-29b and miR-29c in NSCLC tissues were 0.413±0.025，0.609±0.039 and 0.161±0.013，all lower than those in adjacent normal tissues with statistical significance（P＜0.05）. MiR-29a level of NSCLC was positively correlated with miR-29b （r=0.637，P=0.000），and miR-29a levels were positively correlated with miR-29c（r=0.775，P=0.000），and miR-29c levels were positively correlated with miR-29b（r=0.586，P=0.000）. In NSCLC tissues，miR-29s levels were related to TNM stage. Additionally，miR-29a was related to tumor size and differentiation degree，miR-29b was related to the degree of differentiation，and miR-29c was related to tumor size. The median overall survival of NSCLC with higher levels of miR-29a（≥0.354），miR-29b（≥0.586）and miR-29c（≥0.118） were 16.4，16.4 and 16.7 months，better than 11.5，9.8 and 10.5 months of NSCLC with lower levels of miR-29a，miR-29b and miR-29c（P＜0.05）. Conclusion Decreased expression of miR-29s were observed in NSCLC tissue，and they were related to TNM staging and prognosis. They may be related to the occurrence and development of NSCLC，which have a certain value in the diagnosis and assessment of NSCLC.