Background: Long non-coding RNA ADAM metallopeptidase with thrombospondin type 1 motif, 9 antisense RNA 2 (ADAMTS9-AS2) was recognized as a novel tumor suppressor and plays an important role in the initiation and progression of malignant behavior in human cancers, although its plasma expression and clinical value in patients with non-small cell lung cancer (NSCLC) remain unknown. We aimed to analyze the diagnostic role of ADAMTS9-AS2 and cytokeratin 19 fragmentation antigen (CYFRA 21-1) in NSCLC. Methods: The present study included 80 control subjects, 80 patients with benign lung lesion and 80 NSCLC patients. The expression of ADAMTS9-AS2 in the tissue and plasma was detected by a real-time polymerase chain reaction. Serum CYFRA 21-1 was analyzed using an enzyme-linked immunosorbent assay. Results: In comparison with benign lung lesion and controls, tissue and plasma ADAMTS9-AS2 expression were significantly down-regulated in NSCLC (p < 0.001). Decreased ADAMTS9-AS2 expression was associated with TNM stages in NSCLC patients (p < 0.001). Up-regulation of CYFRA 21-1 was reported among NSCLC patients and it was associated with TNM staging. Tissue and plasma ADAMTS9-AS2 expression levels were the predicting factors for NSCLC and they both correlated negatively with CYFRA 21-1 levels. Plasma ADAMTS9-AS2 levels had a significant positive correlation with their tumor tissue levels. Plasma ADAMTS9-AS2 showed a higher sensitivity (95%) and specificity (99.1%) in the diagnosis of NSCLC than CYFRA 21-1 (61.3% sensitivity and 60% specificity). Conclusions: Our results suggested that decreased plasma ADAMTS9-AS2 expression might act as a novel non-invasive tumor biomarker in NSCLC diagnosis. Furthermore, plasma ADAMTS9-AS2 might predict aggressive tumor behavior.