In order to investigate the underlying mechanisms for food intake regulation of Cynoglossus semilaevis, one of the key feeding factors-orexin A-was produced in vitro in prokaryotic expression system and its possible role in feed intake regulation was explored using in vitro incubation method of hypothalamus gland. The matured peptide fragment of orexin A was amplified and subcloned into the prokaryotic expression vector-pET32a to successfully construct orexin A/pET32a recombinant plasmid which was highly expressed in E.coli BL21 after being induced by IPTG with special fusion polypeptides containing His6 at their N-terminus. The orexin A fusion protein was mainly expressed in supernatant liquid with molecular weight of 24.9 kDa, and the fusion protein products maximally accounted for 52.8% of the whole bacterial protein post 6h induction with 1 mmol/L IPTG at 32℃. Western blotting analysis indicated fusion protein had the antigenicity to His6 antibody. Supernatant after crushing, was purified using Ni²⁺-NTA affinity chromatography, then the purified protein with molecular weight of 24.9 kDa was obtained. Incubating hypothalamus with recombinant expression orexin A of Cynoglossus semilaevis indicates that orexin A can significantly influence expression of NPY mRNA, orexin A mRNA and secretion of NPY peptide in hypothalamus. Therefore, the obtained recombinant orexin A protein has biological activity in the present study. The present results would be helpful for better understanding the roles of orexin A in feeding regulation and development of high-efficient feeding promotion additives for aquaculture of Cynoglossus semilaevis.