The need for the discovery of new antibiotics and solving the antibiotic resistance problem requires rapid detection of antibiotics, identification of known antibiotics, and prediction of antibiotic mechanisms. The bacterial lux genes encode proteins that convert chemical energy into photonic energy and lead to bioluminescence. Exploiting this phenomenon, we constructed a lux-based bioluminescence system in Staphylococcus aureus by expressing lux genes under the control of stress-inducible chaperon promoters. When experiencing antibiotic stress, these constructed reporter strains showed clear bioluminescence response. Therefore, this bioluminescence screening system can be used for the detection of antibiotics in unknown chemical mixtures. Further analysis of bioluminescence response patterns showed that: (1) these bioluminescence response patterns are highly antibiotic specific and therefore can be used for rapid and cheap identification of antibiotics; and that (2) antibiotics having the same mechanism of action have similar bioluminescence patterns and therefore these patterns can be used for the prediction of mechanism for an unknown antibiotic with good sensitivity and specificity. With this bioluminescence screening assay, the discovery and analysis of new antibiotics can be promoted, which benefits in solving the antibiotic resistance problem.