Abstract: Flavobacterium davisii， a pathogen of columnaris disease， significantly impacts the tilapia aquaculture industry. In order to diagnostic columanris disease in Nile tilapia quickly and exactly， in the report， the recombinant plasmid containing TonB gene （iron complex protein， TonB） was constructed， the specific PCR primers， TonB-F/R， targeting TonB， were designed， and a reliable absolute quantitative PCR detection method was established and was used for detecting bacterial loads in tilapia infected with the pathogen. The results demonstrated that the specific primers， TonB-F/R， did not cross-react with different genotypes of Flavobacterium or other common pathogens in tilapia. The sensitivity of the established absolute quantification method was 100 times higher than that of conventional PCR， with excellent repeatability and stability. The bacterial loads in the gill tissues of tilapia infected with F. davisii were significantly higher than that in other tissues， which was consistent with the infection characteristics of the disease. In summary， the real-time qPCR detection method offers advantages of rapidity， high sensitivity， and strong specificity， which can perform the accurate assessment of F. davisii infection levels. The method is valuable for the prevention and control of columnaris disease.