The aim of the present study is to develop a surface plasmon resonance sensor for the detection of vitamin B2, vitamin B9, and vitamin B12 in food samples by using the molecular imprinting technique. The vitamin B2, vitamin B9, and vitamin B12 imprinted and the non-imprinted surface plasmon resonance sensor chip surfaces were characterized by using contact angle measurements, atomic force microscopy, ellipsometry, and Fourier transform infrared-attenuated total reflectance. The real-time detection of vitamin B2, vitamin B9, and vitamin B12 was analyzed by using aqueous solutions in the concentration range of 0.01 ng/mL - 10 ng/mL for vitamin B2, 0.1 ng/mL - 8.0 ng/mL for vitamin B9, and 0.01 ng/mL - 1.5 ng/mL for vitamin B12. The limit of detection values was calculated as 1.6×10^-4 ng/mL for vitamin B2, 13.5×10^-4 ng/mL for vitamin B9, and 2.5×10^-4 ng/mL for vitamin B12, respectively. Selectivity experiments were performed by using vitamin B1 and vitamin B6. The reproducibility of surface plasmon resonance sensors was investigated both on the same day and on different days for four times. Validation studies of the prepared surface plasmon resonance (SPR) sensors were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).