In the present study, an immunosensor was designed to assess bovine mastitis at the earliest by quantifying leukotoxin M/F′-PV (LukM/F′-PV), a potent neutrophil targeting and beta-barrel pore-forming toxin secreted by bovine strains of S. aureus. Polyclonal antibodies to the recombinant LukF (rLukF) component of LukM/F′-PV was raised and purified by affinity chromatography. Further, anti-rLukF antibody was used to design a classical ELISA detection system in which we obtained 1000 ng ml⁻¹ LOD. Considering the drawbacks of the classical detection system, antibodies were functionalized to gold nanoparticles of a large surface plasmon band providing an opportunity to design immunoassays based on nanosurface energy transfer (NSET) from dye to gold nanoparticles (GNPs). In an experimental set up, rLukF was incubated with functionalized GNPs, fluorescein isothiocyanate (FITC)-labeled secondary antibodies were added, and fluorescence quenching was monitored as a function of toxin concentration. With this method, leukotoxin was detected in the 100–0.1 ng ml⁻¹ range, with a LOD of 0.1 ng ml⁻¹ and R² = 0.9908. In addition, the above proposed assay was performed to detect toxins in spiked and field samples with 89–95% recovery. Thus, the proposed method overcomes the major drawbacks of ELISA systems and can provide a window for sensitive detection of toxin before onset of mastitis.