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In the process of anaerobic fermentation of Propionibacterium freudenreichii to produce vitamin B12 deoxyadenosylcobalamin (ADO), the intermediate adenosylcobinamide (CBI) and 5,6-dimethylbenzimidazole (DMB) are combined to produce the product ADO. At present, there is lack of detection means for vitamin B12. To solve the problem, taking CBI as the research goal, the metabolic regulation method is used to improve the addition time of DMB, and the effects of different DMB adding time on the growth of bacteria and ADO synthesis are studied. Under the conditions of batch fermentation, the CBI synthesis amount and ADO yield of Propionibacterium are determined, and the growth curve of the bacteria is drawn. It is found that the inhibitory effect of DMB on the growth of the cells is not obvious, but the unit cell yield could be inhibited. The fermentation cycle is 120 h, and the optimal addition time is 90~100 h. Compared with 60 h, the addition of DMB can increase the fermentation unit by 59.08%. The method is simple, rapid, efficient, and has a certain guiding effect on the study of fermentation regulation of vitamin B12.