We report a novel fluorescence-based immunoassay which enables qualitative detection of the Staphylococcus aureus Thermonuclease (TNase) enzyme, thus providing confirmation of the presence of the S. aureus bacterium in vitro. The biomedical problem of chronic wound healing and the continuing emergence of antibiotic-resistant species is addressed in the development of a detection system capable of the rapid, real-time assessment of bacterial load and diversity. The use of bispecific antibodies (BsAb) provides integration of the molecular detection and signal response components of a standard immunoassay due to steric hindrance-mediated release of prebound fluorescent reporter molecules upon specific binding of TNase to adjacent sites. Rhodamine and fluorescein-labeled hemocyanin from Megathura crenulata (KLH) were prepared as effective immunoconjugates containing a sensitive fluorescent reporter moiety. BsAb that both specifically quenched the fluorescence of the reporter conjugate and bound the TNase target antigen were produced using cell fusion techniques. Assays were then performed to analyze the properties attributable to the steric hindrance-mediated release of the fluorescent reporter molecules upon adjacent TNase binding. This was performed by monitoring the intensity of fluorescence emission of the immunogenic reporter conjugate released into an aqueous environment at 578 and 520 nm, respectively.