Staphylococcal enterotoxin B (SEB) is one of many toxins produced by the Gram-positive bacterium Staphylococcal aureus. While SEB is known as the causative agent of certain food poisonings it is also considered a biological Select Agent. Thus, rapid and accurate identification of SEB during either surveillance or in response to a biothreat is critical to the mitigation of the suspect agent. This report presents an improved method for the detection of SEB based on a SEB-specific, two-antibody system where one antibody was bound to a magnetic bead particle while the other was labeled with Alexa fluor 647. The assay consisted of one incubation period for 30 minutes where all reagents necessary to detect SEB were included. Using this assay 100 pg of recombinant purified SEB, as well as SEB from the culture supernatant of several strains of methicillin-resistant S. aureus were detected with fidelity. This assay presents improvements over current assays in terms of a combination of the reduction in assay time length, assay sensitivity, ease of use, and application to automated high-throughput analysis. Additionally, this assay can be easily modified to detect a wide range of proteins and whole organisms.