Florescence in situ Hybridization(FISH) was used to analyze the amount and spatial distribution of Karst mountainous soil sulfate-reducing bacterium(SRB), 16S rRNA specific probe used with DAPI total cell staining and dilute technology, FISH can in situ detect the amount and spatial distribution of SRB in soil profile efficiently. The results showed that, there were sulfate-reducing bacteria in each layer of soil profile. The minimum cell numbers was (0.8±0.4)×107cells/g and maximum cell numbers was (6.2±1.3)×107cells/g , the average was(2.7±1.2)×107cells/g. FISH is a rapid and effective detection technology in the research of spatial and temporal distribution of SRB in environment that can qualitative and quantitative analysis SRB in the soil simultaneously.