Abstract: The fermentation condition of Bifidobacterium animal B04 isolated from centenarians’ intestine, were optimized for bacteriocin production. The bacteriocin inhibitory activity (AU/mL) was used as evaluation index and quantified against L. monocytogenes ATCC 35152. Firstly, the effects of the single factors (inoculum, initial pH, fermentation temperature and time) on bacteriocin production from strain B04 were examined. Based on these single factor tests, response surface methology with three variables of initial pH, fermentation temperature and time was employed to optimize the fermentation condition for bacteriocin production. Results showed the optimal conditions were inoculum 1%, initial pH 6.3, fermentation 29h at 36℃. Under these optimal conditions, the bacteriocin inhibitory activity was increased by 2.37 times and reached up to 3479.25AU/mL. Finally, the consistent results between the prediction and experiments indicated the established model in this study is feasible.