Objective To evaluate a real-time fluorescence quantitative PCR of dengue virus by detection of key indicators in the dengue mouse model. Methods Appropriate specific primers and probes were selected, the molecular method was used to prepare the plasmid standard, and the PCR system and reaction conditions were optimized. Then, the sensitivity, specificity, and stability of the real-time fluorescence quantitative PCR method were evaluated. Finally, this method were tested by using serum and tissue samples from dengue virus-infected mice. Results One-step real-time fluorescence quantitative PCR was performed successfully. The method was sensitive, and the lowest detection limit was 49. 6 copies/ μL. The method was specific, and non-specific amplification was not observed. The method repeatability was good, the standard deviation of sample Ct values was less than 0. 5, and the CV was <5%. The test result of samples from the dengue virus-infected mice were in line with experimental expectations. Conclusions The qRT-PCR method can be used for quantitative detection and evaluation of dengue virus in a mouse model.