The enhancement by means of magnesium salt of the production of a thermal stable nuclease of Vibrio vulnificus with a recombinant Escherichia coli was investigated. Supplementation of the medium with a proper amount of magnesium salt was found to be critical for enzyme production. Cultivation of the recombinant strain in LB medium resulted in a nuclease yield of 62 U/mL. Supplementation of the LB medium with 50 mM MgCl2 increased the yield to 380 U/mL. Increasing the cell concentration could enhance the nuclease yield provided that a sufficient amount of magnesium ion was present in the medium. However, an overdose of magnesium salt had an adverse effect on enzyme production, where the type of magnesium salt was an influential factor. A nuclease yield of 3750 U/mL was achieved by supplementing LB medium with additional carbon and nitrogen sources, and with a proper concentration of MgSO4.