In this study, media optimization by statistically designed experiments stimulated an increase in the intensity and stability of bioluminescence of Photobacterium phosphoreum KCTC 2852 during continuous cultivation. The Plackett-Burman design method selected two components (NaCl, yeast extract) among nine components of the seawater complete (SC) medium. The response-surface design method calculated the optimum concentration of NaCl (31.1 g/l) and yeast extract (5.1 g/l). In the optimized medium, bioluminescence per cell increased twofold. During continuous cultivation in a stirred tank reactor, bioluminescence decreased rapidly in the SC medium, whereas the optimized medium could maintain bioluminescence for more than 80 h, resulting in an increased bioluminescence. Upon contact with 0.1 ppm Hg2+, a sharp decrease in bioluminescence was observed, which was reproducible in the measuring chamber. This shows the possibility that P. phosphoreum could be cultivated continuously with enhanced bioluminescence stability and that on-line water toxicity could be monitored continuously.