The aim of the present study was to detect microRNA (miRNA) signatures in advanced non-small cell lung cancer (NSCLC), and to study the association between miRNA expression levels in serum and tissue. A cohort of patients who had previously been diagnosed with advanced NSCLC was enrolled in the present study. miRNAs associated with prognosis, which had previously been detected in early stage NSCLC samples, were measured in the serum of the patient groups using a cross‑validation method. In addition, serum miRNAs associated with progression‑free survival (PFS) were detected in paired fresh tissue samples, in order to analyze the correlation between serum and tissue expression levels. A risk‑score analysis was used to develop a four‑miRNA signature to predict PFS. miR‑1, miR‑30d, miR‑221 and miR‑486 were identified as having a significant correlation with PFS in advanced NSCLC. miR‑221 and miR‑486 exhibited significant positive correlations between serum and tissue expression. Furthermore, overexpression of miR‑221 and reduced expression of miR‑486 increased cell proliferation, migration and invasion in vitro. In conclusion, the miRNA signature identified in the present study may be considered an independent prognostic factor of PFS in advanced NSCLC. In addition, the expression levels of miR‑221 and miR‑486 were significantly correlated between serum and tissue. miR‑221 was identified as an oncogenic risk factor, whereas miR-486 exerted protective effects against cancer cell proliferation, migration and invasion.