In this work, a ratiometric fluorescent probe for Vitamin C (VC) detection was constructed based on water-soluble silicon nanoparticles (SiNPs), o-phenylenediamine (OPD) and MnO2 nanosheets. Firstly, bright blue fluorescent SiNPs with high quantum yield were synthesized by a hydrothermal method using (3-aminopropyl)triethoxysilane (APTES) as silicon source and guanine as reducing agent. Then, MnO2 nanosheets with a wide absorption band were prepared. In the sensing system, MnO2 nanosheets play three crucial roles: fluorescence quencher of SiNPs, oxidation OPD to yellow fluorescent 2,3-diaminophenazine (oxOPD) with a emission at 560 nm, and VC recognition. The SiNPs/OPD/MnO2 probe possessed two obvious emission peaks at 465 nm and 560 nm under 405 nm excitation. With the addition of VC, MnO2 nanosheets were reduced, and the fluorescence quenching ability and oxidase-like properties were weakened, thus restricting the production of oxOPD and recovering the fluorescence of SiNPs. Therefore, with the increase of VC concentration, the fluorescence (FL) intensity of the sensing system increased at 465 nm while decreased at 560 nm. Based on this, a ratiometric fluorescence method was established for VC detection by using the FL intensity ratio (F560/F465) as signal readout. The detection range was 0.5-40 μM with a limit of detection (LOD) of 0.42 μM. Moreover, the proposed method demonstrated good practicability for VC detection in VC tablets, VC chewable tablets and VC injection with satisfactory recoveries.