Dengue fever, a mosquito-borne viral disease has become a major worldwide public health problem with a dramatic expansion in recent years. Similarly, Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. There is neither an antiviral therapy nor any effective vaccine available for dengue. Early diagnosis plays a crucial role to forecast an early warning of epidemic and to undertake effective vector control measures for dengue and Japanese encephalitis. Envelope domain III (EDIII) protein is involved in binding to host receptors and it contains speciﬁc epitopes that elicit virus neutralizing antibodies. The objective of the present work is to develop high yield and scalable production process for recombinant dengue and Japanese encephalitis envelope domain III proteins in Escherichia coli, purification process to achieve high purity and biologically active protein as well as their characterization for use as diagnostic reagent in enzyme linked immunosorbent assay (ELISA) and possible vaccine candidate molecule. Expression of EDIII proteins of JE and Dengue viruses was carried out in recombinant Escherichia coli. Developments of cost effective and simple culture media as well as appropriate culture conditions are generally favorable for large scale production of recombinant proteins. Optimization of culture media was carried out for enhanced production of EDIII protein in E. coli. Laboratory scale batch fermentation process in E. coli was developed using optimized media and culture conditions. Furthermore, fed-batch fermentation process was also developed in optimized medium. Expression of this protein in E. coli was induced with isopropyl β-D-thiogalactoside. The protein was overexpressed in the form of insoluble inclusion bodies (IBs). Cells were disrupted using sonicator or agitator bead mill and IBs were purified. For diagnostic studies, the protein was purified under denaturing conditions using affinity chromatography. The affinity chromatography purified protein was used as an antigen to develop enzyme linked immunosorbent assay (ELISA) to detect antibodies in infected serum and CSF samples. In order to take this vaccine candidate for further studies, recombinant EDIII protein was produced employing a pilot scale fermentation process. Recombinant EDIII protein expressed as inclusion bodies was solubilized in the presence of urea and renatured by oncolumn refolding protocol in the presence of glycerol. A three-step purification process comprising of on-column refolding with affinity chromatography, ion-exchange chromatography (IEX) based on salt, and IEX based on pH was developed. The purity of the recombinant EDIII protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA. Biological function of the refolded and purified EDIII protein was confirmed by their ability to generate EDIII-specific antibodies in mice that could neutralize the virus. These findings suggest that recombinant EDIII protein is highly immunogenic and elicit high-titer neutralizing antibodies. These results establish the application of these proteins to be used for the diagnosis of JE and Dengue virus infection or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant viral proteins.