Background Vitamin B12 status is assessed primarily by measuring total serum B12 using competitive binding methods. The lack of availability of a standard material and high-level reference measurement procedure affect the trueness of B12 results; this results in variation between methods. This study aimed to determine the reference intervals for vitamin B12 on three routine analytical platforms. Method A prospective reference population of healthy individuals was recruited according to the IFCC CRIDL criteria. Vitamin B12 samples were measured on Roche, Beckman and Siemens analytical platforms. Results In total, 300 adult subjects were recruited; the central 95th centile values for B12 for Roche (190–678 ng/mL) and Siemens (181–562 ng/mL) analytical platforms were in a close agreement. Beckman DXi, however, showed a significantly lower reference limit (110–562 ng/mL). All reference intervals are in keeping with previously published data but some are not in agreement with manufacturer provided reference interval. Conclusion As the quality of the reference intervals plays a significant role in clinical outcome, it is of great importance that laboratories use a method-specific reference interval and if possible, locally derived reference intervals until further method standardization occurs.