Leptin, a 16 kDa protein hormone encoded by the obesity gene (ob), is secreted by adipose tissue and essential for regulating various physiological processes, such as fat metabolism, feeding, reproduction, and immunity. Seriola aureovittata, prized for its flavor and high nutritional content, often experiences excessive fat accumulation under artificial farming conditions because of spatial constraints in farming facilities and the provision of fresh fish as feed. This fat accumulation can adversely impact their growth and quality. A recombinant leptin protein for S. aureovittata was constructed using a prokaryotic expression vector to investigate the multiple physiological functions of leptin, and its biological activity was verified via intraperitoneal injection. This study provides technical support for further investigation of the physiological regulatory role of leptin in the growth and fat metabolism of S. aureovittata and for developing specialized products for growth and quality control. Total RNA was isolated from the brain tissue of S. aureovittata, and the first strand of cDNA was synthesized. Mature peptide fragments were synthesized based on amino acid sequences encoded by lepa and lepb in S. aureovittata. Using the prokaryotic expression vector pQE30, recombinant plasmids lepa/pQE30 and lepb/pQE30 were constructed and transformed into Escherichia coli M15. Following induction with 0.5 mmol/L IPTG at 37 ℃ for 4 h, the expressed proteins were validated for their expected sizes via western blot analysis, revealing clear bands at approximately 17.9 and 17.3 kDa. This confirmed that the recombinant proteins exhibited antigenic activity and were specifically recognized by 6×His antibodies. Further purification of the target proteins via a Ni²⁺-NTA affinity chromatography column yielded purified LepA and LepB recombinant proteins, with purity verified at ＞90% and endotoxin levels ≤ 1 EU/μg. Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified proteins showed distinct bands at approximately 17.9 kDa and 17.3 kDa, aligning with the expected size of the recombinant proteins, thereby confirming the effective purification of the LepA and LepB fusion recombinant proteins. The concentrations of the purified LepA and LepB recombinant proteins were measured at 0.2 and 0.3 mg/mL, respectively, using a protein quantification kit. In the intraperitoneal injection experiment, four groups were established: a control group (0.9% saline solution) and experimental groups with concentrations of 0.05, 0.1, and 0.2 μg/kg. Each group consisted of 18 fish reared in a 1 m³ water tank. Each fish received an intraperitoneal injection of recombinant leptin protein at a dose of 1 μL/g body weight. At 6, 12, and 24 h after the first injection, brain tissues from six randomly selected fish were used to detect biological activity. After injecting three concentration groups (0.05, 0.1, and 0.2 μg/kg) of LepA and LepB proteins at different time points (6, 12, and 24 h), the expression of lepa and lepb genes in the brain was either upregulated or downregulated, indicating that the recombinant proteins LepA and LepB possessed biological activity. These results provide technical support for further research into the physiological functions and regulatory mechanisms of leptin in the growth and development of S. aureovittata