In this study, we investigated safety and efficacy of a low temperature immunization protocol with NNV in red spotted grouper, Epinephelus akaara and long tooth grouper, Epinephelus bruneus and seven band grouper, Hyporthodus septemfasciatus. Further, growth rate between immunized and na?ve fish was evaluated during the experiment to check side effect of immunization. Three grouper species were immunized by immersion method with live NNV at 105.0 TCID50/mL at 16.5˚C for 30 min and reared for 120 days at natural sea water temperature. To evaluate growth rate, total length and wet weight was measured 7 times after immunization. Immunized three grouper species were challenged by intramuscular inoculation with NNV at 104.2 TCID50/100 ?L/fish. Immunization at low temperature with live NNV did not show any clinical symptoms of infection, mortality and inhibition of growth. After challenge, cumulative mortality of na?ve seven band grouper, red spotted grouper, long tooth grouper were 45, 10, 20 %, respectively. However no mortality was observed at immunized groupers. Thus, it was demonstrated that immunization at low temperature with live NNV are able to protect three different species of groupers without inhibition of growth.