The ease of manufacture, distribution, and resiliency of Bacillus anthracis endospores make this bacterium an attractive bioweapon to terrorists. Rapid identification of Bacillus anthracis endospores is crucial for implementing a response strategy in the event of an attack. Because current detection methods are based on time-consuming culture and fluorescence techniques, an optical biosensor is being developed to identify an unknown endospore in real-time without fluorescent labels. This study was conducted to enumerate, detect, and differentiate endospores of B. atrophaeus, B. pumilus, and B. thuringiensis as simulants for B. anthracis, and to perform preliminary testing of the biosensor. Fluorescence analysis showed polyclonal B. atrophaeus antibodies bound all three organisms, monoclonal B. atrophaeus antibodies cross-reacted with B. pumilus, and monoclonal B. thuringiensis antibodies bound only B. thuringiensis. The limit of fluorescence-based detection was approximately 30 endospores. The biosensor showed comparable sensitivity, with results complete within 30 minutes. The results of this study demonstrate that the biosensor is able to detect endospore-antibody binding without fluorescent labels in timely fashion, suggesting that this system holds promise for rapid identification of B. anthracis endospores.