Objective To detect three kinds of enterovirus in mouse cecal contents by nucleic acid detection techniques, and analyze the result of nucleic acid detection compared with serological tests to determine the applicability of nucleic acid detection for the health monitoring of mice. Methods Samples of mouse cecal contents from our laboratory were randomly selected to extract nucleic acids. Real?time PCR was used to detect the three enteroviruses MHV, Reo?3, and MNV in these samples. Nucleic acid detection result and serological antibody test result were correlated and analyzed. Results Among the 272 samples, the nucleic acid?positive rates for MHV, Reo?3, and MNV were 17. 3%, 18. 8%, and 16. 9%, respectively. From the detected amounts of nucleic acid of MHV and Reo?3 in samples, they were mostly in a low?copy state. Correlation analysis showed that the nucleic acid detection result were not completely correlated with the result for detecting antibodies to the three viruses. Conclusions It is possible for mice as experimental animals to carry the three viruses MHV, Reo?3, and MNV. As a supplement to serological antibody detection technology, nucleic acid detection can be used as a highly sensitive method to monitor the health of experimental animals.