ABSTRACT. Abnormal expression of microRNA-135a (miR-135a) is closely associated with oncogenesis. However, the relationship between serum miR-135a levels and the clinical parameters and prognosis of non-small cell lung cancer (NSCLC) remain unclear. The study aimed to investigate the clinical significance of serum miR-135a expression in patients with NSCLC. miR-135a expression was analyzed by realtime PCR and its correlation with NSCLC was determined by various statistical methods for 104 NSCLC patients and 40 healthy volunteers. The serum miR-135a level was significantly lower in NSCLC patients than in healthy control subjects (P < 0.01), and was closely related to distant metastasis (P < 0.015), lymphatic metastasis (P = 0.000), TNM (tumor node metastasis) stage (P = 0.001), and pathological stage (P = 0.021) of NSCLC. The five year overall survival was significantly lower in patients with low miR-135a expression than that in patients with high serum miR-135a levels (P = 0.010). Multivariate analysis showed that serum miR-135a level could be treated as an independent risk factor for NSCLC prognosis (P = 0.011). In conclusion, the serum miR-135a level was downregulated in NSCLC patients, and was associated with poor prognosis. Additionally, it can be used as a biomarker for NSCLC prognosis.