Bangia fuscopurpurea is a traditional mariculture crop having high nutritional value, eicosapntemacnioc acid (EPA) production, and protein content. As an intertidal species, it can tolerate drastic changes in abiotic factors such as temperature, hydration, and light radiation; however, genomic information on the evolutionary aspect and mechanism of EPA enrichment in B. fuscopurpurea and the role of EPA in cold tolerance in this species remain elusive. We conducted transcriptome profile analysis in B. fuscopurpurea to investigate the biological functions of genes associated with resistance to various environment factors. We identified 41,935 unigenes that were assembled and applied to public databases to define their functional annotation (NR, GO, KEGG, KOG, and SwissProt). We further identified genes that encoded key enzymes in EPA biosynthesis; five paralogous genes encoding delta5 desaturase were detected in B. fuscopurpurea. Fatty acid profiling and gene expression analysis of B. fuscopurpurea grown under cold stress were simultaneously performed. The EPA content was increased by 29.8% in the samples grown at 4°C, while the total amount of fatty acids remained unchanged. Moreover, all the EPA biosynthesis-related desaturase and elongase genes were upregulated under cold stress. Thus, we hypothesized that diverse EPA biosynthesis pathways and significant increase in gene copy numbers of fatty acid desaturases, together with the concomitant elevation in the transcriptional level of genes associated with fatty acid metabolism, lead to EPA accumulation and subsequently affect membrane fluidity, contributing to cold stress resistance in B. fuscopurpurea. Our findings not only provide a fundamental genetic background for further research in B. fuscopurpurea, but also have important implications for screening and genetic engineering of algae and plants for EPA production.