PURPOSE: To afford the methane fermentation process with a new added value by a specific treatment of a liquid extract into which vitamin B12 derivatives have been extracted from the bacteria sludge produced by methane fermentation process to separate the title substances in high efficiency at low cost from the above sludge.

CONSTITUTION: Firstly, a liquid extract into which vitamin B12 and its derivatives (VB12s) have been extracted from the bacteria sludge produced by methane fermentation process is brought into contact, at pH ≤5, with an adsorptive resin incapable of ion exchange consisting of a styrene-divinylbenzene copolymer to effect adsorption of the VB12s. Secondly, the VB12s are eluted from said resin with an organic solvent followed by removing the organic solvent from the resultant elute. The elute is then made alkaline and brought into contact with an anion exchange resin as hydrophilic matrix allowed to contact with an alkaline solution in advance to effect adsorption of the VB12 to said resin. Finally, a salt solution is brought into contact, under an alkaline condition while successively increasing its concentration, with the above anion exchange resin, thus effecting the separation (desorption) of the VB12.