Tgf2 transposase (Tgf2-TPase), a hAT transposase from goldfish, plays an important role in fish transgenic applications. Previously, the production of the recombinant Tgf2-TPase protein required rigorous fermentation at low temperatures (22 °C) and early log phase induction (OD600 = 0.3–0.4) in Rosetta 1 (DE3) Escherichia coli lines. In order to better express the Tgf2-TPase and detect its enzyme activity, 83 rare codonsin Tgf2-TPase were optimized and designated Tgf2-TPase83. The expression results showed that the soluble recombinant Tgf2-TPase83 was highly expressed at 30 °C and was inducible at an OD600 of 0.5–0.6 in the same prokaryotic expression system. After purification by affinity chromatography, Tgf2-TPase83 with codon optimization had higher enzyme activity than the Tgf2-TPase control. Comparison of different preservation methods (freeze-drying at −80 °C, storage in 20%-glycerol, 8%-sucrose, 4%-mannitol), revealed storage of Tgf2-TPase83 in glycerol helped to preserve its DNase digestion activity. Furthermore, size exclusion chromatographysuggested that the purified Tgf2-TPase83 could recognize and bind to DNA probescontaining a terminal inverted repeat (TIR) and a subterminal repeat (STR) sequence of the Tgf2 transposon. Overall, the results showed that optimizing the 83 codons of Tgf2 transposase can simplify the fermentation process and improve the enzyme activity. We propose that the production of the Tgf2-Tpase83 protein in a soluble and active form could provide an alternative tool for genetic modification of fish.