In order to investigative the effect of different neutralizers and their feeding time on culture of Bifidobacterium bifidum BB01, and pH, OD and viable count of B. bifidumBB01 in the medium in different time were measured. The results indicated that the NaOH solution was the optimum neutralizer compared with the others, and feeding time of the neutralizer to B. bifidum BB01 was 13 hours after inoculation. Furthermore, the OD value and viable count reached maximum at 21h (OD value= 1.667) and 20h (viable count: (3.52±0.046) ×10⁹ CFU/mL) after the NaOH solution was added to the medium, respectively. In addition, the maximum OD value implied that the logarithmic phase of B. bifidum BB01 was delayed compared with the control and the viable count were 29.26% larger than the control group. The result of the study provides a method and an important basis for improving the viable counts of B. bifidum BB01.