<p>Rapid and accurate detection of <i>Staphylococcus aureus</i> (<i>S. aureus</i>) is significant to ensure food safety and curb the spread of the epidemic. Conventional single-mode optical sensors for detecting <i>S. aureus</i> are susceptible to interference from complex sample matrices. We propose a label-free fluorescence/colorimetric dual-mode sensor for <i>S. aureus</i> through its intrinsic alkaline phosphatase (ALP) activity. The biosensor is based on carbon dot-loaded manganese dioxide nanosheets (CDs@MnO₂) nanocomposites. In the presence of <i>S. aureus</i>, ALP catalyzed the hydrolysis of ascorbic acid 2-phosphate magnesium ester, yielding ascorbic acid. The resulting ascorbic acid suppresses the sensor’s oxidase-like activity and mitigates the inner filter effect, thereby reducing the absorbance of substrate and restoring fluorescence. This method achieved a low detection limit of 2&#xa0;cfu mL^‒1 and was successfully applied to milk, milk powder, pork, and lettuce samples, yielding recoveries of 90.7%‒110% with relative standard deviations below 5.9%.</p> Graphical abstract <p></p>