Vitamin B₁₂ (VB₁₂) is one of the essential B complex vitamins, it is a water soluble nutrient and a medicine. Herein we developed, a novel but straightforward method for the selective and sensitive quantitative determination of VB₁₂ based upon measuring the fluorescence quenching of functionalized CdS quantum dots (QDs) in aqueous media. The mercaptopropionic acid (MPA) functionalized CdS QDs were directly synthesized from cadmium chloride and sodium sulfide in aqueous media by a chemical method. The detection mechanism involves photoinduced charge transfer from CdS QDs towards VB₁₂ at the surface of the CdS QDs resulting in the fluorescence quenching of CdS QDs followed by nonradiative fluorescence resonance energy transfer. Under the optimized conditions, the relationship between the fluorescence intensity of the CdS QDs and VB₁₂ concentration was linear in the range of 5.0–100 μg mL⁻¹ with a limit of detection of 6.91 μg mL⁻¹. The selectivity experiment indicated excellent selectivity for VB₁₂ over a number of interfering species. Moreover the method was successfully applied in the determination of VB₁₂ in complex matrices, namely blood serum, urine and pharmaceutical formulations (multivitamin injection), without sample pretreatment. The accuracy and reliability of the method was further ascertained by recovery studies via the standard addition method with percentage recoveries in the range of 97.15%–99.49% being obtained. In addition, the probable fluorescence quenching mechanism of the CdS QDs induced by VB₁₂ is also discussed.