Objective: To compare the effects of clinical application of chemiluminescence and real-time, fluorescence-based quantitative PCR in the detection Epstein-Barr virus (EBV). Methods: The data of chemiluminescence and real-time fluorescent quantitative PCR from 97 patients who were suspected of being infected with EBV from January 2016 to January 2019 in our hospital were analyzed. The specific stage of EBV infection was analyzed, and the differences in results of the two detection methods were compared. Results: Chemiluminescence method was used to detect EBV infection during the active phase. The sensitivity of the chemiluminescence method was 76.7% (56/73) and the real-time quantitative PCR method was 90.4% (66/73). There was a statistical difference between the two detection methods (P<0.05). Conclusion: There was no statistical difference in positive predictive values between the chemiluminescence method and the real-time, fluorescence-based quantitative PCR method in the detection of EBV infection, but the sensitivity of chemiluminescence method is slightly lower than the real-time quantitative PCR method. It is noteworthy that chemiluminescence method is convenient and fast while the real-time, fluorescence-based quantitative PCR method is more accurate, which can provide a more accurate reference for clinical treatment.