Turbot (Scophthalmus maximus) is a main breeding marine fish species in northern China, but its aquaculture industry is seriously threatened by bacterial diseases. The turbot cell line will provide effective tools for the study of turbot immunity and basic biology. In this study, a continuous passaged Scophthalmus maximus gill (SMG) cell line was established through primary culture and subculture, and the optimal culture conditions were determined. The culture medium consists only of a basal medium L-15, fetal bovine serum (FBS) and antibiotics without supplementing growth factors and other additives. Its turbot-derivation was verified by chromosomal analysis and 18S rRNA sequencing. We also confirmed that pEGFP-C1 plasmid could transfect SMG cells. After lipopolysaccharide (LPS) stimulation, the transcriptome of SMG cells changed. The results of gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the differentially expressed genes were significantly enriched in immune-related signaling pathways such as Toll-like receptor signaling pathway and RIG-I-like receptor signaling pathway. Taken together, we established an easy-to-culture cell line from turbot gill, which provides a convenient tool for studying turbot diseases and immune mechanisms.