The aim of this study was to set up a high level prokaryotic expression of heat shock protein 90(HSP90) of Exopalaemon carinicauda in E. coli Rosetta. The HSP90 gene of E. carinicauda was cloned into prokaryotic expression vector pET-30a, which was confirmed by double-endonuclease digestion and DNA sequencing. The recombinant vector was transformed into E. coli Rosetta and was induced to express under different temperatures, durations, IPTG concentrations and OD600. The expressed product was identified by SDS-PAGE and an 82.7 kD protein (determined by mass spectrometry) was found. The expression level varied under different conditions. The optimal expression was achieved under induction conditions of 1 mmol/L IPTG,37℃, OD600=0.58, and 7 h.