The intake and monitoring of vitamin B12 levels are highly essential to maintain a healthy body condition. Herein, we report bismuth oxide nanoparticles (Bi2O3 NPs) facilitated electrochemical sensing of vitamin B12 with admirable sensitivity, selectivity, and low detection limit. A facile hydrothermal method was adopted for synthesizing Bi2O3 NPs and their physico-chemical properties were analyzed using various techniques. The electrochemical sensing of vitamin B12 was validated through cyclic voltammetry and differential pulse voltammetry techniques. The sensing results illustrate that the modified electrode exhibits a wide linear range between 10 nM and 275 nM with a low detection limit value of 3.66 nM. In addition to the enhanced electrocatalytic behavior, the Bi2O3 NPs have also enabled the discrimination of vitamin B12 from various potential interfering molecules (even adding a 5-fold concentration). The developed sensor also exhibits better stability, selectivity, and reproducibility towards the target analyte. Finally, the fabricated electrode was applied for the determination of vitamin B12 in serum, and plasma samples, and the results were highly satisfactory with recovery values of 98.4 % to 99.8 %.