Abstract: Recombinant E. coli was used as a host to explore the biosynthesis of Hyaluronic acid (HA) in Gram-negative strain using the optimized medium. In addition, measurement of HA molecular weight produced by the recombinant strains in shake-flask and 2 L stirred tank bioreactor (batch and fed-batch cultivation) were compared too .In this study, the hasA and hasE genes expressed together in a plasmid in recombinant E. coli strains were successfully produced HA using glucose as a carbon source under aerobic condition. Production of 127 mg/L HA was observed in a batch fermentation process with the highest HA molecular weight (70056 Da) using strain sAE in 2 L stirred tank bioreactor with controlled DO at 30%. The fed-batch culture with constant feeding of glucose was favorable for cell growth and the highest OD600 achieved at 1.923. However, the HA biosynthesis and molecular weight in fed-batch culture were lower than that observed in the batch culture.