Viral infections in the aquaculture industry present a significant challenge that requires further investigation. Emerging evidence suggests that long noncoding RNAs (lncRNAs) play a crucial role in regulating various biological processes, including immune responses. However, most studies have focused on model organisms, leaving a gap in knowledge about non-coding RNAs in response to viral hemorrhagic septicemia (VHS) in rainbow trout. In this study, we investigated the profile of non-coding RNAs (ncRNAs) and their targeting-related subjects in the spleen of rainbow trout challenged with the VHS virus. We explored their potential roles in immune defense responses through transcriptomic analysis of both virus-treated and control groups. Our analysis identified 4207 lncRNAs from 7753 transcripts, with many categorized as potentially novel isoforms (code = j) and intergenic lncRNAs (code = u). The expression analysis demonstrated the impact of viral infection on the expression levels of lncRNAs and their target genes. The results showed 182 lncRNAs and 1326 mRNAs with differential expression. We identified pairs of DElncRNA-DEmRNA interactions, such as "TP63-MSTRG.62, IL10-MSTRG.9783, IL10-MSTRG.9784, TK1-MSTRG.22826, and GLI1-MSTRG.17881" as cis interactions, while DOCK10-MSTRG.30015, MSTRG.36357-LOC101268921, and MSTRG.34190-ENPP2 exhibited trans interactions. Additionally, key signaling pathways, including the PPAR signaling pathway, Toll-like receptor signaling pathway, and p53 pathway, were revealed as critical responders to viral infection linked to lncRNAs. We also introduced quantitative trait loci (QTLs) associated with these lncRNAs, underscoring their potential regulatory roles in viral stress responses. Overall, our findings provide novel insights into the molecular mechanisms underlying viral infections and lncRNA-mediated host immune responses.