Abstract: Objective：To express fructosyl amino acid oxidase (FAOX) in prokaryocytes for establishing an enzymatic method to determine glycosylated hemoglobin A1c (HbA1c) and evaluate its methodology. Methods：The recombinant plasmid pET32a(+)/FAOX was constructed and transformed into E.coli Rosetta (DE3) to obtain recombinant FAOX after expression and purification. The glycated hexapeptides was used as substrate to determine the activity of recombinant FAOX. The enzymatic method for determination of HbA1c was established and its methodology was evaluated. Results：The recombinant FAOX was highly expressed in E.coli Rosetta (DE3). The relative molecular weight of FAOX was about 65 000 as shown on SDS-PAGE and its specific activity was 22 U/mg. The coefficients of variation (CV) for within run, between run and inter day assay of the enzymatic method in determining HbA1c were all less than 5%. The recovery rates of HbA1c at low value (5.1%) and high value (15.8%) were 98.1% and 98.9% respectively. No interference of total bilirubin (＜220 μmol/L), triglyceride (＜10.8 mmol/L) and vitamin C (＜500 mg/L) on the determination was observed. The results of the enzymatic method showed close correlation with those of HPLC assay and HA-8160 automated glycated hemoglobin analyzer. Conclusion：FAOX could be highly expressed in E.coli Rosetta (DE3). The enzymatic method developed in this study should be satisfied for clinical requirements in HbA1c determination.