Abstract: In order to investigate the spatial distribution of Thiobacillus denitrificans in the intensive pond system and the relationship between the number of Thiobacillus denitrificans and some physicochemical indicators in the water body, columnar sediments samples and stratified water samples water were collected, and quantitative analysis of Thiobacillus denitrificans in the samples were done with the method of most probable number (MPN), at the same time, such physicochemical indicators in the water samples as the pH, the redox potential, the sulfide, the nitrate nitrogen and the nitrite nitrogen were analyzed. The results showed that: the numbers of Thiobacillus denitrificans in the sediments and water samples all decreased with the depth increased. For the sediments, the numbers decreased from 1.4×105 strains per gram (dry weight) to 9×102 strains per gram (dry weight). And for the water samples, the numbers decreased from 4.5×103 strains per milliliter to 1.4×102 strains per milliliter. And through correlation analysis, a positive correlation between the numbers of Thiobacillus denitrificans and the nitrate nitrogen, and a negative correlation between the numbers of Thiobacillus denitrificans and the sulfide were found, the reason for which might be that the spatial distribution of the nitrate and the sulfide were strongly affected by the distribution of dissolved oxygen and the nitrate induced the assimilative nitrate reductase in the cell.