Vitamin B12 is classified as being amongst the hydrophilic vitamins, and plays a crucial role in mortal physiology, and hemoglobin formation and function. It also promotes anti-inflammatory action and moderates risk from viral infection. This paper established an electroanalysis method to quantify vitamin B12 in commercially available supplements by designing a sensor using methylene blue and zinc oxide nanoparticles on a glassy carbon electrode (PMb/ZnO NPs/GCE) via cyclic voltammetry. This offers a wide linear range with high sensitivity for vitamin B12 detection via differential pulse voltammetry using the Co(II/I) redox pair. Sensor morphology and thickness were studied, as was the pH of the supporting electrolyte. To understand the influence co-species, an interference study was also conducted. Under optimized conditions, the redox peak current for Co(II/I) pair at -0.8 V vs. Ag/AgCl, with the linear relationship Ip = 0.0673x + 0.3449, r = 0.9942, showed a linear quantification range for vitamin B12 concentrations of 0.099–69.51 μM. The limit of detection was 0.0104 µM. Repeatability, sensitivity, and stability were evaluated. The PMb/ZnO NPs/GCE electrode developed was successfully applied to the determination of vitamin B12 in commercially available supplements. The recoveries obtained were in the range 97.1-104% for the injection and 95.9-103.3% for the tablet. The results obtained herein compared favorably with those from the current standard quantification via UV–vis spectrometry.