Expression and clinical significance of micro-RNA-21, PTEN and p27 in cancer tissue of patients with non-small cell lung cancer (NSCLC) were investigated. In this study, cancer tissue and adjacent tissue specimens from 230 patients with NSCLC were collected from thoracic surgery department in Hubei Cancer Hospital from March 2010 to February 2016. The expression of miRNA-21, PTEN and p27 in cancer tissue and adjacent tissue of patients with NSCLC was detected by RT-PCR. Combined with clinical information, the correlation among miRNA-21, PTEN, p27 and clinical features of NSCLC was analyzed. The expression of miRNA-21, PTEN, p27 in NSCLC was significantly lower than that in adjacent tissue by RT-PCR (P<0.05). There was no significant difference in age, sex and course of disease (P>0.050), but there were differences in smoking, lymph node metastasis, TNM stage and differentiation degree classification (P<0.050). By comparing the 3-year survival rate in the group with high and low expression of miRNA-21, PTEN and p27, it was found that the 36-month survival rate of patients with high expression of miRNA-21 was 85.19% (P<0.05), and of patients with low expression of miRNA-21 it was 95.90% (P<0.05). The 36-month survival rate of patients with high expression of PTEN was 85.59% (P<0.05), of patients with low expression of PTEN it was 94.96% (P<0.05) and in patients with high expression of p27 it was 84.91% (P<0.05). The 36-month survival rate of patients with low expression of p27 was 94.35% (P<0.05). The survival rates of miRNA-21, PTEN and p27 low expression groups were significantly higher than those of high expression groups (P<0.05). In conclusion, the expression of miRNA-21, PTEN and p271 in cancer tissue of NSCLC patients was low. The three indexes have good diagnostic efficacy based on ROC curve analysis, and are expected to be excellent indexes for early clinical diagnosis and prognosis of NSCLC.