In 1998, the orexin was independently discovered by two different laboratories, and it was latterly proved to have wide functions in feeding promotion, energy balance regulation, growth and reproduction in vertebrates. It is secreted and released from hypothalamus and can encode two peptides, orexin A and orexin B. They exhibit high conservation in sequence during evolution, and the prior research verified that they both have important functions in feeding regulation and energy metabolism in mammals and fish. Cynoglossus semilaevis is an important economic farmed flatfish and a favorite table fish in China. Its farming industry is expanding, however, the endocrine mechanisms underlying growth and feeding actions remain limited. To explore the possible endocrine mechanisms for food intake regulation of C. semilaevis, the matured peptide fragment of orexin B was amplified and subcloned into the prokaryotic expression vector pET-32a to successfully construct orexin B/pET-32a recombinant plasmid. The recombinant plasmid was high-efficiently expressed in E. coli BL21 strains after being induced by IPTG with special fusion polypeptides containing His6 at their N-terminus. The molecular weight of obtained orexin B polypeptide is 21.14 ku and maximally accounted for 43.5% of the whole bacterial protein post 6h induction with 1.0 mmol/L IPTG at 37℃. Western blotting analysis indicated that the recombinant polypeptides had the antigenicity to His6 antibody. The recombinant protein could be purified using Ni²⁺-NTA affinity chromatography. In vitro incubation of hypothalamus with recombinant expression orexin B of C. semilaevis indicated that it could promote the secretion and release of NYP peptide from hypothalamus, and upregulate the expression level of NPY mRNA. Meanwhile, the recombinant orexin B protein also could upregulate the expression level of orexin mRNA in hypothalamus at low concentration. All these verified that the obtained recombinant orexin B protein has biological activity in the present study. The present results would be helpful for better understanding the role of orexin B in feeding regulation and development of high-efficient feed additive for farming industry of C. semilaevis.