Long non-coding RNAs (lncRNAs) represent a class of RNA molecules that do not encode proteins. Generally studied for their regulatory potential in model insects, relatively little is known about their immunoregulatory functions in different castes of eusocial insects, including , a notoriously invasive insect pest. In the current study, we used , an entomopathogenic fungus, to infect the polymorphic worker castes (Major and Minor Workers) and subjected them to RNA sequencing at different intervals (6, 24, and 48 h post-infection (hpi)). Comprehensive bioinformatic analysis identified 5719 (1869 known and 3850 novel) lncRNAs in all libraries. Genomic characteristics analysis showed that lncRNAs exhibited structural similarities with lncRNAs from other eusocial insects, including lower exon numbers, shorter intron and exon lengths, and a lower expression profile. A comparison of lncRNAs in major and minor worker ants revealed that several lncRNAs were exclusively expressed in one worker caste and remained absent in the other. LncRNAs such as , (6 h), , (24 h), , and (48 h) were only present in major worker ants, while lncRNAs such as , (6 h), , (24 h), , and (48 h) were only detected in minor workers. Additionally, we performed real-time quantitative PCR and experimentally validated these findings. Functional annotation of -acting lncRNAs in major worker ants showed that lncRNAs targeted genes such as , , , , etc. In contrast, apoptosis and autophagy-related genes were identified as targets of lncRNAs in minor ants. Lastly, we identified several lncRNAs as precursors of microRNAs (miRNAs), such as miR-8, miR-14, miR-210, miR-6038, etc., indicating a regulatory relationship between lncRNAs, miRNAs, and mRNAs in antifungal immunity. These findings will serve as a genetic resource for lncRNAs in polymorphic eusocial ants and provide a theoretical basis for exploring the function of lncRNAs from a unique and novel perspective.