A suspension array-based multiplexed immunoassay was developed for rapid, sensitive, specific, and simultaneous detection of multiple biothreat-associated agents in powder samples. The 5-plexed immunoassays using sets of 9-plexed coupled fluorescent beads were employed to simultaneously detect five representative biothreat agents, including B. anthracis spore, Y. pestis, SARS-CoV, staphylococcal enterotoxin B (SEB) and ricin from a single powder sample and the feasibility for field samples was demonstrated by both blinded and standard laboratory trials. The detection sensitivity and dynamic range for the five biothreat agents from different powders might be varied depending on the nature of the powder and the feature of the contaminating agent. The limit of detection for Y. pestis, B. anthracis spores, SEB, ricin, SARS-CoV N protein in milk powder was 20 cfu, 111 cfu, 110pg, 5.4 ng and 2 ng per test respectively. Compared to conventional ELISA method, the suspension array has a higher sensitive ability, and can detect five biothreat agents simultaneously with high reproducibility.