A species-specific oligonucleotide sequence was screened from the third variable domain of 16S rRNA gene of Thiobacillus denitrificans. It confirmed that the sequence had the high speciality by amplifying the activated sludge DNA in high simultaneous biological removal of sulfate and nitrate process and further determining the sequences of 16S rRNA gene clone library. The Cy3-labeled species-specific probe hybridized well with activated sludge and obtained good picture under the conditions of 48°C, 35% formamide deionized, and 100 mmol/L NaCl. ImageJ software demonstrated that T. denitrificans counted for 15% of bacteria in the sludge. The proposal of species-specific primer and probe for T. denitrificans would play an important role in disclosing its temporal and spatial distribution, structure dynamics and realtime quantity.