The objective of the present study was to obtain the optimum proportion of the carbon source and prebiotics for Bifidobacterium bifidum BB01 by the central composite design (CCD). The effect of carbon source (lactose) and two prebiotics (inulin and fructooligosaccharides) on the BB01 were observed by measuring the OD₆₀₀ value, pH value and the viable counts at 18h. The final optimized concentrations of carbon source and prebiotics were: lactose 1.6%, inulin 0.26%, and fructooligosaccharides 0.22%. The result indicates that the growth of B. bifidum BB01 shows an significant increase in the optimized culture medium (p < 0.05), the OD₆₀₀ value reached 1.434 at 18h, which increased 6.58% compared to the control. And the viable counts of B. bifidum BB01 increased 24.36% and reached (2.17±0.06) ×10⁹cfu/mL. The results show that the optimization of the carbon source and prebiotics using CCD in this study is workable and necessary.