In biological warfare, it is important to identify biological agents for proper treatment. We focused on developing a real-time RT-PCR kit that can detect multiple species of biological agents. AccuPower(R) Biothreat Real-Time RT-PCR Kit(v3.0) could detect Bacillus anthracis, Yersinia pestis, Vibrio cholerae, Francisella tularensis, Salmonella typhi, Rickettsia prowazekii, Variola virus, Hantaan virus, Yellow fever virus, Brucella spp., Shigella dysenteriae in a single reaction. The results showed that the kit was verified to be able to detect at least 0.005 ng of nucleotide and 10,000 CFU/ml of bacteria. Therefore, the kit is expected to be used as a rapid and sensitive detection kit for 11 species of biological agents within 2 hours.