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Background: The aim of this study was production of a recombinant antigen from envelope protein of dengue virus, in order to achieve high level expression in soluble form. Therefore, the possibility of envelope protein domain III expression in bacterial host was studied. Methods: Multiple sequence alignment for domain III sequences was carried out using Megalign software. The Modeller and Optimizer software were used for protein structure prediction and sequence optimization. After gene synthesis and subcloning in the pET21a expression vector, the optimization of recombinant protein expression was carried out in Escherichia coli. Ni-NTA chromatography columns were used for protein purification. The efficiency of protein expression was analyzed using electrophoresis and western blotting methods. Findings: A consensus sequence for domain III of dengue virus type 2 envelope protein was provided. The structural properties of target protein were predicted using bioinformatics methods. For high expression level, the sequence of EDIII2 gene was optimized for appropriate codon usage and GC content. The coding gene sequence was approved by sequencing and enzymatic digestion. For disulfide bound formation in protein structure, the Origami (DE3) expression host was used. At the final step, expression of target protein was optimized, and a high concentration of recombinant protein was achieved in soluble form. Conclusion: The results of this study showed that the optimization of domain III sequence led to obtain high concentration (20 mg/l) of produced protein. This expression system can be used for production of dengue virus envelope protein domain III. [ABSTRACT FROM AUTHOR]