Abstract: Objective：To establish experimental animal Salmonella detection method based on a high-throughput sequencing technology, and apply it to detect Salmonella in animal experiment. Methods: Extracted DNA samples from mice excrement, designed universal primers for 16S rDNA, 23S rDNA, 16S-23S rDNA, 23S~5S rDNA region, gyrB preferred area respectively, tested and analyzed each primer, determined best amplification conditions and build a database, distinguish 42 samples of Salmonella by Illumina high-throughput sequencing technology and evaluate the specificity and stability of the method. Results The species preferred regions of Salmonella was gyrB gene region, the primers for gyrB gene was FP5’-AACCACCGCAATCA GACCTT3’, FP5’-AGCCACGAAACCTTCACYA-3’. The primers were optimized and determined, through high-throughput sequencing and sequence analysis can detect the 42 samples in a very small amount of Salmonella, detection method is stable, high sensitivity, limit of detection can reach 0~102 CFU. Conclusion This experiment using high-throughput sequencing technology, established a complete detection to detect Salmonella in experimental animals. This system can detect trace amounts of salmonella in experimental animals, and the detection method is stable and high sensitivity, which could be used to other kinds of pathogenic microorganism detection.