Pathogenic bacteria cause disease outbreaks and threaten human health, prompting the research on advanced detection assays. Herein, we developed a selective molecular imprinted aptasensor for sensitive and prompt quantitation of Staphylococcus aureus (S. aureus) bacteria. The aptasensor was constructed by immobilization of aptamer on gold nanoparticles modified magnetic nanoparticles (apt-AuNPs@ Fe₃O₄). A functional monomer (o-phenylenediamine, o-phen) was electro-polymerized on the surface of the as-synthesized nanocomposite in the presence of a template (S. aureus). After removing S. aureus, the formed imprinted sites were available to extract pathogenic bacteria from complicated matrices. The surface morphology of the as-fabricated nanocomposites was characterized using different spectroscopic and electrochemical methods. Moreover, we thoroughly evaluated factors affecting the synthesis and determination procedures. The molecular imprinted aptasensor exhibited a wide linear range of 10¹–10⁷ CFU mL⁻¹ with a Limit of Detection, LOD (signal to noise = 3) of 1 CFU mL⁻¹. The aptasensor detected S. aureus in milk, conduit water, and apple juice samples with good recoveries % and satisfactory relative standard deviations (RSDs %) values.