Abstract: Through the method of ultraviolet mutagenesis to Bifidobacterium longum,a strain was screened with resistance ability to oxygen,the strain was named L80. Using L80 as the test strain,the optimum culture medium was determined by single factor,response surface and flow and feeding method. High density culture medium optimization formula was( mass fraction) soy peptone 4.03%,yeast extract powder 2.02%,glucose 0.95% and stachyose 1.59%.The optimal fed- batch cultivation was: the sugar concentration was kept in 6.0 ~8.0 g / L,p H6.5,and the final viable bacteria number was reached( 1.21 ± 0.03) × 10¹⁰ cfu / m L.