A simple, sensitive, and selective method based on the fluorescence quenching of BSA-stabilized gold nanoclusters (BSA-AuNCs) for rapid determination of vitamin B12 is developed. The fluorescence emission was measured at λ_ex/λ_em 370/617 nm in phosphate buffer solution of pH 7.4, and the experimental variables and possible interference were studied. Under optimized conditions, the proposed method allows the determination of vitamin B12 over the range 160.0 ng mL⁻¹ to 38.5 μg mL⁻¹ with a correlation coefficient of 0.998 and a limit of quantification of 100.0 ng mL⁻¹. The proposed method was successfully applied to commercial injection dosage forms. The recovery of the method is in the range of 97.7–102.0% and the relative standard deviation is in the range of 2.0%–5.9%.