Strategies for enhancing the production of Vibrio vulnificus nuclease using a recombinant Escherichia coli were explored. Using a batch culture, a maximum nuclease yield of 3500 U/mL was obtained with a complex medium characterized by high concentrations of yeast extract, tryptone and magnesium ion. The formation of acetate during fermentation was considered to be detrimental to the nuclease production. Both dissolved oxygen (DO) control and glucose feeding could suppress acetate formation, thus enhancing nuclease production, where the former was found to be more effective. Using a pH-stat fed-batch culture with the DO level maintained above 15% air saturation and with intermittent addition of nutrients, a nuclease yield of 12000 U/mL was achieved.