Objective To evaluate enzyme immunostaining technique for detection of F1 antigen of Yersinia pestis in rodents. Methods Visceral organ specimens of 266 mice infected with virulent Y. pestis and 207 control rodent specimens were detected by horseradish peroxidase labeled plague F1 monoclonal antibody (HRP-F1McAb) enzyme immunostaining technique, and in comparison with the RIHA and RGICA methods．Results Coincidence was 98.52% between HRP-F1McAb enzyme immunostaining technique and RIHA, Kappa=0.970, and the difference was statistically significant in the positive detection rates (χ²=5.140, P=0.016); Coincidence was 98.50% between HRP-F1McAb enzyme immunostaining technique and RGICA, Kappa=0.901, with statistically insignificant difference in the positive detection rates (χ²=0.250, P=0.625). Sensitivity of HRP-F1McAb enzyme immunostaining technique was 100%, specificity was 97.02%, positive predictive value was 97.14%, negative predictive value was 100%, and Youden index was 0.970． Conclusion The enzyme immunostaining technique is sensitive and specific, fast and simple in detection of plague F1 antigen. It is a valuable detection technique in early and rapid diagnosis of plague in rodents．