The 2001 anthrax alarm in the US raised concerns about the Nation's preparedness to the threat of bioterrorism, and the demand for early warning systems that might be used in the case of a biological attack continues to grow. Here we develop an ultra-sensitive rapid detection method for B. globigii (BG) spores, the simulant of B. anthracis (BA) spores. BG spores were detected by a bead-based sandwich immunoassay with fluorescence detection. Paramagnetic Dynal^® beads were used as a solid support, primary antibody was attached to the beads by streptavidin-biotin coupling and the secondary antibody had an alkaline phosphatase (AP) enzyme label. Enzymatic conversion of fluorescein diphosphate (FDP) to fluorescein by AP was measured in real time with λ_ex = 490 nm and λ_em = 520 nm. The assay was linear from 2.6 × 10³–5.6 × 10⁵ BG spores mL⁻¹, and the detection limit was 2.6 × 10³ spores mL⁻¹ or 78 spores. All reagent concentrations and incubation times were optimized. The assay time from the moment the spores were introduced to the system was 30 min, and real-time fluorescence detection was done in less than 1 min. Formation of the BG spores–capture beads complex was confirmed by environmental scanning electron microscopy (ESEM). BG spores were detected successfully when doped into Cincinnati tap water to demonstrate the applicability of the developed method to detect the spores in non-buffered media.