Triple TaqMan real–time PCR method was developed to simultaneously detect sulfonamides–resistance genes Sul1, Sul2 and Sul3, the three pathogenic bacteria in aquatic animals in the study. Three pairs of specific primers and fluorogenic–labeled probes were designed and synthesized in accordance with the above target genes using software Primer Express 3.0. The reaction system and procedure were optimized, as well as the detection sensitivity, specificity, repeatability and clinical application of the method. Results showed that the method had a wide quantitative range from 1×101 to 1×108 copies per reaction, which presented a good linear relationship in its standard curve. The triple real–time PCR method had a high specificity in detecting mixed DNA of Sul1, Sul2 and Sul3, but not those DNA from other bacteria and viruses; it also had a high sensitivity to the detection limit low to 10 copies per reaction for the puri?ed recombinant plasmids of Sul1, Sul2 and Sul3. The variation coef?cients of the established method were less than 2%. Sulfonamides–resistance genes of 72 clinical isolates were analyzed using triple real–time PCR method and they were compared to drug sensitive test, the coincidence rate could reach to 94.2%. The entire detection could be completed within 2 h. In conclusion, the triple TaqMan real–time PCR method developed in the present study could be applied to the rapid detection and molecular epidemiology survey in sulfonamides–resistance genes Sul1, Sul2 and Sul3 of clinical pathogenic strains isolated from aquatic animals.