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This work aimed at developing a ratiometric fluorescence sensor for sensitive and accurate detection of ascorbic acid (AA) based on oxidase-mimicking activity of CoOOH nanoflake. CoOOH nanoflake with oxidase-like activity was prepared by using CoCl2 as precursor, NaClO as oxidant through a sonication method. The as-synthesized CoOOH nanoflake was characterized by transmission electron microscopy (TEM), UV–vis absorption, X-ray diffraction (XRD), and Fourier transform infrared (FT-IR) spectra. Control experiments were used to demonstrate the feasibility for constructing the radiometric fluorescent sensor using the as-synthesized CoOOH nanoflake. Single-factor experiments were conducted to optimized the optimal concentration of CoOOH nanoflake and incubation time for AA detection. The results showed that the as-synthesized CoOOH nanoflakes exhibited typical hexagonal plate shape, and other characterization results also were consistent with those in previous works. The optimal concentration of CoOOH nanoflake was 39.1 μmol/L, and the optimal incubation time was 25 min. Under the optimal conditions, it was found that F428/F568 linearly correlated with AA concentration from 0.5 to 10.0 μmol/L with correlation coefficients of 0.9965. The regression equation was y=0.79416x+0.37917 (R2=0.9965) with a limit of detection of 303 nmol/L (RSN=3). The recoveries from fruit juice beverage samples spiked with varying concentrations of AA were in the range of 88.0%~115.9%, and analytical results of the sensor were correlated well with those of national standard method of China. These results indicated that the ratiometric fluorescent sensor had excellent selectivity and accuracy for AA detection, creating a new avenue for rapid detection of AA.