Objective To establish a simple, stable, specific and sensitive method for detection of Tyzzer's organism by reverse dot blotting (RDB). Methods Primers and specific probes were designed according to the conservative sequence of Tyzzer 16S rDNA. The forward primer was labeled with biotin. The reverse dot blotting method was established followed by PCR amplification. The specificity and sensitivity of this method were determined. Next, 41 mice and 38 rats were examined by RDB, ELISA and IFA. Results The RDB method showed a high specificity, and in the testing of the 79 laboratory animals, its limit of detection was 4.5 ng/μL. Compared the results of ELISA and IFA, its consistence with ELISA was 100% and the positive rate was 7.59% (6/79), the consistence with IFA was 92.4% (73/79), and the positive rate was 0%.Conclusions An accurate, sensitive and specific method in combination with PCR and RDB in detection of Tyzzer's organism is established in this study.