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Ascorbic acid (AA) detection in biological sample and food sample is critical for human health. Herein, a MnO2 nanosheet (MnO2-NS)-based ratiometric fluorescent nanosensor has been developed for high sensitive and specific detection of AA. The MnO2-NS presents peroxidase-like activity and can oxidize non-fluorescent substrate of o-phenylenediamine (OPDA) into fluorescent substrate, presenting maximum fluorescence at 568 nm (F568). If MnO2-NS is premixed with AA, the MnO2-NS is then decomposed as Mn2+ by AA, decreasing the fluorescent intensity of F568. Meantime, AA is oxidized as dehydroascorbic acid (DHAA), which can react with OPDA to generate fluorescent substrate. A new fluorescence response is found at 425 nm (F425). The dual fluorescent responses can be excited with a universal excitation wavelength, simplifying the detection procedure. With F425/F568 as readout, limit of detection for AA reaches as low as 10.0 nM. Satisfactory recoveries are found for AA detection in serum and diverse beverages. The ratiometric strategy significantly eliminates false-negative and false-positive results, providing a cost-effective, rapid, and reliable way for AA detection in real sample.