In order to further study the function of gene SIRT3 in Takifugu rubripes, the full-length open reading frame (ORF) of gene T. rubripes SIRT3 (trSIRT3) was amplified from total RNA in their liver tissue through reverse transcription-polymerase chain reaction (RT–PCR). The recombinant expression vector pET32a/SIRT3 was generated from the prokaryotic expression vector pET32a (+) derived from sub-cloned ORF, then, it was transformed into E.coli Rosetta (DE3). As the result of IPTG induction, SIRT3 was successfully expressed in E.coli Rosetta (DE3). Result of sequence analysis showed that the ORF of SIRT3 was 1 263 bp and encoded with 420 amino acids. The prokaryotic expression system of recombined vector pET–32a/SIRT3 was successfully constructed. From the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the recombinant protein had an approximate molecular weight of 66 000, which was consistent with the theoretical molecular weight. The fusion protein with tag His, was mainly detected in the supernatant.