Abstract: Fish nocardiosis is a common fish disease that seriously harms the global aquaculture industry. Nocardia seriolae is one of the main pathogens of fish nocardiosis. In this study, the specific gene fragments with intraspecific conservation and interspecific variation were screened by comparing and analyzing the whole genomes of 17 reported N. seriolae strains, and specific primers were designed to establish a specific PCR method for the detection of N. seriolae. The designed primers can only amplify the specific fragments of N. seriolae, no PCR fragments showed in other bacteria, and the sensitivity of PCR detection is up to 80 pg/μL, 8.6 CFU/μL. The PCR method was used to detect the tissues of hybrid snakehead with N. seriolae infection or negative control, the results showed that the positive fragments could only be amplified from the tissues of N. seriolae-infected fish. In this study, based on the comparison and analysis of the whole genome data of different strains of N. seriolae, the established PCR method is more reasonable and reliable than the previous molecular detection method. This PCR method can provide a rapid, accurate and sensitive detection for N. seriolae.