According to the 16S-23S rRNA gene sequences of Nocardia seriolae available in GenBank, a pair of primers was designed for establishing a SYBR Green I real-time fluorescence quantitative PCR method. It had a good linear relationship between initial templates and Ct values, and the correlation coefficient of the standard curve was 0.998. The sensitivity analysis showed that the developed SYBR GreenⅠreal-time PCR could detect 10^-6 μg/μL DNA. The specificity assay showed that negative control and the other bacteria such as Aeromonas hydrophila, Escherichia coil, Staphylococcus aureus, could not be detected by this PCR. Through the epidemic seasons, 16 fish tissue sample, 4 water samples and 8 fish bait tissue samples were detected by the real-time PCR assay. Results showed that 7 out of those samples were positive, which had good agreement (100%) with bacteriological analysis by isolation and culture. It was able to diagnose the clinically diseased fishes, and to recognize the carrier of N. seriolae as well. It reflected application value on early prevention and control of disease. The results showed that the developed SYBR GreenⅠreal-time PCR assay had the advantages of specificity, sensitivity, rapidity and quantitativity, and was able to be applied to the clinical diagnosis of N. seriolae in fish.