<p>The growing burden of <i>Staphylococcus aureus</i> (<i>S. aureus</i>) infections, especially those involving antibiotic-resistant strains necessitates the development of advanced detection technologies that are rapid, selective, and adaptable to real-world conditions. In the present work, amine-functionalized sulfur quantum dots (NH₂-SQDs) conjugated with <i>S. aureus</i> specific antibodies have been explored for highly sensitive and specific fluorescence detection of respective bacteria. To this end, NH₂-SQDs were synthesized using solvothermal methods followed by their conjugation with polyclonal anti-<i>S. aureus</i> antibodies using EDC/NHS chemistry to develop immunofluorescent conjugate probe, NH₂-SQDs-Ab. The so-formed immunofluorescent probe was characterized using different spectroscopic and microscopic techniques to establish the successful synthesis and antibody conjugation. Further, the NH₂-SQDs-Ab was investigated for the precise identification of <i>S. aureus</i> through targeted immunofluorescence. The results demonstrated that NH₂-SQDs-Ab probe is highly specific towards <i>S. aureus</i> even in complicated matrices containing other interfering bacteria. The immunosensor demonstrated a strong and proportional fluorescence response to different <i>S. aureus</i> concentrations, thereby, achieving a detection limit of 10&#xa0;CFU/mL over a broad linear range of 5× 10¹ to 5 × 10⁴&#xa0;CFU/mL, along with outstanding reproducibility (<i>R</i>² = 0.9975). Furthermore, the developed immunofluorescent probe also showed outstanding recoveries in real-world samples, which further validates its potential for useful applications in environmental monitoring and food safety.</p> Graphical Abstract <p></p>