Bacillus anthracis represents a serious threat of biological weapons, and there is an urgent need to develop a rapid, simple and sensitive assay to detect B. anthracis spores in environmental samples. Commercial lateral flow immunoassays for detection of B. anthracis spores are easy to perform but exhibit very low sensitivity. This study aims to develop a sensitive and simple dipstick test, based on agglutination reaction between B. anthracis spores and antibody-coated magnetic particles, to detect spores of this pathogen. To this aim, factors affecting the test performance including antibody, nitrocellulose membrane type, incubation time and pH of reaction between detection conjugate and sample, and concentration of Tween-20 were optimized. Under the optimal condition (monoclonal antibody to Anthrax Spore Specific 23A-14G9, UniSart® CN95 nitrocellulose membrane, 30 minutes of incubation, pH 9.0, and 1% Tween-20), the developed test could detect B. anthracis spores with the limit of detection of 6 × 104spores/mL, and showed no cross-reactivity to B. thuringiensis, B. cereus, B. subtilis, B. clausii, Yersinia bercovier và Yersinia rohdei and E. coli. When applied to detect B. anthracis spores in simulated starch samples, the developed assay has a detection limit of 107spores/g starch. This new immunoassay is particularly suitable for screening of B. anthracis spores in powdery samples because of its simplicity and improved sensitivity.