A strain of Bacillus subtilis（BS08） was used as feed additive to study the effects on intestinal flora of tilapia， especially on the adherence， colonization and breeding capacity of spoilage bacteria from intestinal flora of tilapia during ice storage after being fed with Bacillus subtilis. At the same time， the effects of BS08 on delaying spoilage of iced-storage tilapia was also studied. Four treatments were grouped by feeding with Bacillus subtilis and adding spoilage bacteria （Shewanella and Pseudomonas） in water or not. The influences of Bacillus subtilis and spoilage bacteria on intestinal flora of iced storage tilapia from four treatment groups were determined by high throughput sequencing. The TVB-N and K values were used as the corruption index of iced-storage fish. Bacillus subtilis showed the ability to maintain the diversity of the intestinal flora of tilapia and repress the changes of the intestinal flora of tilapia. The degree of colonization of Shewanella in the intestine is greater than that of Pseudomonas. Bacillus subtilis could colonize in the intestine of fish and inhibit the colonization of Shewanella to significantly repress the dominant growth of Shewanella. TVB-N and K value indicated that Bacillus subtilis significantly delayed the corruption of iced-fish.