Recent increasing evidences have linked Omi to neuroprotection and the cellular protein quality control system. However, it is very difficult to produce functionally active Omi to study the role of Omi in vitro. Here, we report a method for mass production of functionally active high quality Omi through heterologous Escherichia coli expression. The heterologously expressed Omi was present mostly in soluble cell fraction rather than insoluble cell fraction as inclusion bodies, but the protein in soluble cell fraction had very poor activity. In this method, low concentration of denaturing agents was added to the lysate for complete solubilization and monodispersion of Omi after lysising Escherichia coli. The solubilized and monodisperesed protein sample was purified by Ni-NTA agarose affinity chromatography at denatured condition. The purified denatured Omi was then refolded by a rapid-dilution in an optimized protein refolding buffer that was designed by systematic variation of parameters favoring the refolding, preventing aggregate formation and influencing the stability of the folding intermediates. Around 20 mg of properly folded Omi was obtained from 1 liter of bacterial culture by this method which is the highest production reported so far. The biological activity was confirmed by its ability to degrade β-casein. This method is a straightforward and efficient purification procedure to obtain a higher yield of proper biologically active Omi and that Omi can be used for the investigation of Omi’s association with neuroprotection in various neurodegenerative diseases.