Long non-coding RNA (lncRNA) plays an important role in the regulation of epigenetics, transcription, and post-transcription. To understand the regulation mechanism of immune response to Vibrio harveyi, we performed lncRNA identification and differential expression analysis from RNA-seq data. We obtained a total of 4 584 lncRNA loci, containing 5 714 transcripts in this study. The comparison of basic characteristics between lncRNA and the coding gene showed that the GC content of lncRNAs was lower than that of coding genes, and single exon genes number was greater than that of coding genes, and the average transcripts length of lncRNAs was longer than that of coding genes, and the average expression of lncRNAs was slightly lower than coding genes. We screened 818, 813, 261 and 140 differentially expressed lncRNAs by differential expression analysis in four comparisons of Cynoglossus semilaevis groups (CsRU vs CsSU, CsRC vs CsSC, CsRC vs CsRU, CsSC vs CsSU), respectively. Additionally, we preliminarily analyzed the expression patterns and the relationship of differential expression of lncRNAs from those four groups. Through co-expression analysis, a total of 14 539 types of interaction relationships were detected between 285 lncRNA and 274 coding genes. We performed GO annotation and screened 7 key lncRNA. The qRT-PCR confirmed that the expression patterns of lncRNAs were consistent with their expression levels bases on transcriptome data. This study provides some of reference data for studying the role of lncRNA in the immune response against V. harveyi.