A ratiometric fluorescence and colorimetry dual-mode nano-biosensor has been established for Staphylococcus aureus (S. aureus) determination. The prepared approaches of Manganese dioxide nanosheets (MnO2 NSs) and carbon dots (BCDs) were facile, efficient and labor-saving and MnO2 NSs-mediated fluorescence quenching and oxidation could amplify detection signals. The dual-mode determination had a broad linear range of 37 ∼ 3.7 × 106 CFU/mL and low detection limits of 9 CFU/mL (ratiometric fluorescence) and 22 CFU/mL (colorimetry). Meanwhile, the method was applied in real samples with recovery ranging of 90 ∼ 102% and RSD < 4.44%, which was an insignificant difference with standard plate counting. The new dual-mode approach of S. aureus possesses the advantages of superior sensitivity, precision, accuracy and specificity. Moreover, the dual-mode nano-biosensor can be adopted in other foodborne pathogens determination by changing corresponding aptamers and provide an enlightenment in monitoring food safety.