The invention relates to the technical field of biology fermentation, in particular to a method for promoting pseudomonas denitrificans to generate vitamin B12. The method for promoting pseudomonas denitrificans to generate vitamin B12 comprises the following steps of through a mother bottle culture medium, a shake flask culture medium and a supplemented medium optimized through massive experiments, under the optimized fermentation condition, production bacterial strains of vitamin B12 are cultured. In 0-39h of the front period of the fermentation process, the temperature is controlled to be at a relatively-low level, namely 26-30 DEG C, the strains are controlled to be at appropriate growth rate and favorable mushroom shapes are maintained. In the middle and later periods of the fermentation process, the temperature of the fermentation system is increased and maintained to be at a relatively-low level, namely 30-34 DEG C, the growth of thalli is promoted, starting and accumulation ofsynthesis of the vitamin B12 are promoted, and further, after the fermentation is finished, the fermentation unit of the vitamin B12 is as high as 198mg/L, so that the yield and the production efficiency of the vitamin B12 are increased, the energy consumption is reduced, and the cost is reduced.