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Fano resonance (FR) is extremely sensitive to extremely small changes in the surrounding environment. We first propose an optical nano-refractive index sensor based on Fano resonance, which is applied to the identification of water-soluble vitamins B1, B5 and B6 and the measurement of the concentration of vitamin B1. The sensor can be used to rapidly identify pure vitamins B1, B5, and B6 at a concentration of 1 g/50 mL at 25 °C based on the relationship between the wavelength shift in the FR line spectrum and the refractive index. This work shows that the sensitivity of the sensor can reach 1327.5 nm/RIU, the sensor can be used to rapidly identify vitamins B1, B5, and B6 through changes in refractive index under certain conditions. Moreover, rapid calculation of vitamin B1 solution concentration is achieved based on the relationship between different concentrations of vitamin B1 solution and their corresponding refractive indexes and wavelength shifts in their FR line spectrums, which is an important step for the application of the designed MIM waveguide structures to the fields of biology, chemistry, and medicine.