<p><i>Staphylococcus aureus</i> (<i>S. aureus</i>), one of the most prevalent foodborne pathogens, poses a persistent global threat to public health by causing severe infections and food poisoning. We present β-cyclodextrin/surfactant inclusion-mediated nucleic acid enrichment strategy synergized with a ratiometric electrochemical biosensor for the robust determination of <i>S. aureus</i>. Unlike commercial kits limited by dilution effects, our method utilizes cetyltrimethylammonium bromide (CTAB) to induce the condensation of nucleic acids from large-volume lysates, followed by a competitive inclusion reaction where β-cyclodextrin encapsulates the hydrophobic alkyl chains of CTAB. This mechanism triggers the efficient release of DNA into a micro-volume, achieving a ~ 12-fold enrichment. The enriched target is subsequently quantified using a ratiometric electrochemical interface equipped with methylene blue (MB) and ferrocene (Fc), where the current ratio (I_MB/I_Fc) serves as an intrinsic self-calibration parameter to eliminate the internal/external disturbances. Under optimized experimental conditions, the developed biosensor demonstrated a wide linear range spanning from 10¹ to 10⁷ CFU/mL, with a low limit of detection of 8 CFU/mL. Moreover, the biosensor achieved reliable quantification of <i>S. aureus</i> in complex real samples, including peach juice and UHT skim milk, confirming its practical applicability. This work establishes a novel and cost-effective electrochemical sensing strategy that couples DNA enrichment and release with ratiometric signal, providing a feasible technical approach for rapid pathogen detection.</p> Graphical Abstract <p></p>