Objective To establish a detection method of Salmonella in laboratory animals based on a high-throughput sequencing technology, and to apply it in detection of Salmonella in laboratory animals. Methods DNA samples were extracted from mouse feces. Universal primers for 16S rDNA, 23S rDNA, 16S-23S rDNA, 23S-5S rDNA region, gyrB preferred area were designed, respectively. Each primer was tested and analyzed to determine the best amplification conditions and build a database. Forty-two samples of Salmonella were assayed by Illumina high-throughput sequencing technology and evaluated the specificity and stability of this method. Results The species preferred region of Salmonella was gyrB gene region. The primers for gyrB gene were FP5'-AACCACCGCAATCAGACCTT3' and FP5'-AGCCACGAAACCTTCACYA-3'. The primers were optimized and determined, through a high-throughput sequencing, and the sequence analysis detected very small amount of Salmonella in the 42 samples, indicating that this detection method is stable, highly sensitive, and the limit of detection reached to 0-10² CFU. Conclusions We have established a complete detection system for detection of Salmonella in laboratory animals based on a high-throughput sequencing technology, This system can detect trace amounts of Salmonella in laboratory animals, and this detection method is stable and highly sensitive, which can be also used in detection of other kinds of pathogenic microorganism in laboratory animals.