Abstract: The peptides encoding ORF domains were synthesized based on the amino acid sequence of lepa and lepb from Cynoglossus semilaevis. The prokaryotic expression vector pEQ30 was used to construct the recombinant plasmids of lepa/pQE30 and lepb/pQE30, which were transformed into Escherichia coli M15 strains and then induced by IPTG to obtain the recombinant proteins of LepA and LepB containing 6 His at the N-terminus. The obtained LepA and LepB polypeptides expressed in form of inclusion bodies had molecular weight of both 16 ku, and the optimum condition for the highest expression of the target proteins were induction by 0.5 mmol/L IPTG at 37 °C for 4 hours. The concentrations of LepA and LepB recombinant proteins in tongue sole were 0.3 mg/mL and 0.25 mg/mL, respectively. The results of Western blot and mass spectrometry analysis indicated that the obtained recombinant proteins of LepA and LepB had correct sequences and immunological activity. The proteins were purified by Ni²⁺-NTA affinity chromatography, and high-purity recombinant proteins of LepA and LepB were obtained. In vitro incubation of hypothalamus with recombinant LepA and LepB proteins from C. semilaevis indicated that they could significantly inhibit the expression of endogenous lepa, lepb and gnrh3 mRNA, which verified that the obtained recombinant proteins have biological activities. The results could help exploration of the physiological role and regulation mechanism of leptin in growth and development of C. semilaevis.