Abstract Background Long non-coding RNAs (lncRNAs) play important roles in lung tumorigenesis. Among different lncRNAs, overexpression of the lncRNA actin filament‐associated protein 1‐antisense RNA 1 (AFAP1-AS1) in lung tumors has been reported in different studies. In the current study, we aimed to investigate the potential value of lncRNA AFAP1-AS1 as a diagnostic biomarker in lung cancer. Ninety samples from patients with lung cancer were collected from Noor-E-Nejat hospital, Tabriz, Iran. The expression of AFAP1-AS1 was assessed using quantitative reverse transcriptase-PCR (qRT-PCR), followed by the ROC curve analysis to investigate the biomarker potency of AFAP1-AS1. Results Our results revealed an upregulation of AFAP1-AS1 in tumor samples as compared to the adjacent non-tumor tissues. We found a significant positive association between AFAP1-AS1 expression and tumor size, as well as tumor stage. Conclusions Our results showed overexpression of AFAP1-AS1 and its capacity as a diagnostic biomarker in lung cancer.