Although the role of long non-coding RNAs (lncRNAs) in key biological processes in animals and plants has been confirmed for decades, their identification in fungi remains limited. In this study, we discovered and characterized lncRNAs inAspergillus flavusin response to changes in water activity, CO2concentration, and temperature, and predicted their regulatory roles in cellular functions. A total of 472 lncRNAs were identified in the genome ofA. flavus, consisting of 470 novel lncRNAs and 2 putative lncRNAs (EFT00053849670 and EFT00053849665). Our analysis of lncRNA expression revealed significant differential expression under stress conditions inA. flavus. Our findings indicate that lncRNAs inA. flavus, particularly down-regulated lncRNAs, may play pivotal regulatory roles in aflatoxin biosynthesis, respiratory activities, cellular survival, and metabolic maintenance under stress conditions. Additionally, we predicted that sense lncRNAs down-regulated by a temperature of 30 °C, osmotic stress, and CO2concentration might indirectly regulate proline metabolism. Furthermore, subcellular localization analysis revealed that up-and down-regulated lncRNAs are frequently localized in the nucleus under stress conditions, particularly at a water activity of 0.91, while most up-regulated lncRNAs may be located in the cytoplasm under high CO2concentration.