Goldfish Tgf2, a transposon of Hobo/Activator/Tam3(hAT) superfamily, encodes active transposase which is capable of mediating gene insertion and mutagenesis in a series of fish species. Therefore, it has great potential in applications such as screening master genes of important traits, functional explanation of genes and fish breeding. For existence of many rare codons in Tgf2 transposase cDNA (Tgf2TP, JN886591), Tgf2TP was optimized based on codon preference of E. coli. The synthesized Tgf2TP was then cloned into pET-28a(+) and transformed into BL21(DE3) cells. After incubation at temperature (37℃) and induction when OD₆₀₀≈0.5, the cells produced high yield of recombinant proteins, which accounted for 8.8% of total proteins in cells and 4.0% of supernatant proteins in lysates. By affinity chromatography purified recombinant protein (70 ku) was obtained, subjected to MALDI-(TOF)/TOF and identified as Tgf2 transposase (Tgf2TP). Then size exclusion chromatography was applied to evaluate DNA-binding activity of Tgf2TP. The results showed that recombinant Tgf2TP recognized and bound specific DNA probes containing sub-terminal repeat sequences of Tgf2, which means start of transposition. The prokaryotic expression system for Tgf2TP not only provided soluble and active transposase efficiently, but also laid a foundation for Tgf2TP used as enzyme tools in a variety of fish biology research.