HbA1c enzymatic method has been considered as a popular method for determination of blood glucose. In this study, the methods for decreasing the formation of Fructosyl Peptide Oxidase (FPOX) inclusion bodies in E. coli and secretion of the enzyme into periplasmic space using PelB signal peptide were investigated. Recombinant FPOX was mainly expressed as inclusion bodies. The inclusion bodies of FPOX were only soluble in urea 8 M, while was not soluble in any of used concentrations of DMSO solvent. By freeze-thawing method, the inclusion bodies was soluble in urea 8 M, but was not soluble in urea 0.5 M. By using the stabilizers on solubility of rFPOX we found that the effect of sorbitol was higher than arginine. The rFPOX was successfully secreted in the periplasmic space using PelB signal peptide that shown the amount of periplasmic protein in shuffle was higher than of BL21. For rFPOX activity, TMB was used as coloring agent for the first time in HbA1c enzymatic method as peroxidase substrate. In conclusion, FPOX inclusion bodies are heterogenous in size and can be decreased using signal peptide. The Freeze-thawing method can be used to solubilize FPOX inclusion bodies at lower concentrations of urea.