Staphylococcal nuclease A (SNA) may be used to produce bacterial ghosts for further inactivation of host bacteria and elimination of residual genetic materials. It is still controversial if SNA without signal peptide can be secreted to extracellular matrix and if fusion with other peptide is required for its function in the cytoplasm of host bacteria. To clarify this dispute, a series of temperature-inducible plasmids carrying SNA alone or SNA fused with partial sequences of λ phage cro gene (cSNA) or Mycobacterium tuberculosis urease gene (uSNA) were constructed and evaluated in Escherichia coli. Results show that the percentages of inactivated E. coli by SNA, cSNA and uSNA after 4 h of induction were 99.9%, 99.8% and 74.2%, respectively. Moreover, SNA and cSNA in the cytoplasm of host bacteria were initially detectable after 30 min of induction, whereas uSNA was after 1 h. In comparison, SNA and cSNA in culture supernatant were initially detectable 1 h later, whereas uSNA was 2 h later. The nuclease activity in the cytoplasm or supernatant was ranked as follows: SNA > cSNA > uSNA, and the activity in the supernatant was significantly lower than that in the cytoplasm. Furthermore, host genomic DNA was degraded by SNA or cSNA after 2 h of induction but not by uSNA even throughout the whole experiment. In conclusion, this study indicates that SNA, cSNA and uSNA expressed in host bacteria all have nuclease activity, the enzymes can be released to culture media, and fusion with exogenous peptide negatively reduces the nuclease activity of SNA.