New methods and technology are needed to detect biological agents that threaten the health of humans and domestic animals. The bacterium Bacillus anthracis, causal agent of anthrax, has been used as a biological warfare agent. Here, we extend the work of Chinowksy et al. (2007) to the detection of a surrogate of B. anthracis, B. globigii (also known as B. atrophaeus, B. subtilis var. niger, B. subtilis var. subtilis) in a mixed sample containing two different species of Bacillus using a portable surface plasmon resonance (SPR) biosensor (SPIRIT 4.0, Seattle Sensor Systems). Two methods (direct capture and antibody injection) were used to determine the limit of detection for spores of B. globigii and to detect spores of B. globigii in a mixed sample containing at least one other Bacillus spp. Spores of B. globigii were detected on freshly coated sensors (not previously exposed to spores) with direct capture at a minimum concentration of 10^7 spores/mL, and with antibody injection at a concentration of 10^5 spores/mL. Spores of B. globigii were also detected when mixed with B. pumilus spores in the same sample at equal concentrations (107 spores/mL) using antibody injection. An SPR method using synthetic miRNA was adapted to the portable SPR unit (SPIRIT), and preliminary experiments suggested that the target sequence could be detected. SPR methods using nucleic acids have an exciting future in the detection of biological agents, such as B. anthracis. With the availability of portable instrumentation to accurately detect biological warfare agents such as B. anthracis, emergency responders can implement emergency protocols in a timely fashion, limiting the amount of people and domestic animals exposed.